InDrop Library Preparation - TruDrop (modified V2) Detailed

Austin Southard-Smith, Alan J Simmons, Ken Lau

Published: 2021-08-04 DOI: 10.17504/protocols.io.9ngh5bw

Abstract

This protocol details the library preparation process of dual-indexed, NovaSeq-compatible, single-cell RNA-Seq libraries from the inDrop V2 platform. The final library structure combines the TruSeq sequencing primers and indices from standard illumina libraries with the inDrop V2 library features such as the cell barcodes, unique molecular identifier, and transcript. As such it is referred to TruSeq-inDrop (TruDrop).

Before start

Prepare the following using RNase and DNase free reagents:

RNA Elution Buffer (RE Buffer)

10millimolar (mM) Tris-HCl, 7.5

0.1millimolar (mM) EDTA

For 5mL:

1Molarity (M) Tris-HCl 7.5: 50µL

0.5Molarity (M) EDTA 10µL

RNAse-free H₂O 4940µL

Aliquot into nuclease-free microfuge tubes. RE buffer can be stored at Room temperature.

DNA Elution Buffer (DE Buffer)

10millimolar (mM) Tris-HCl8.0

0.1millimolar (mM) EDTA

For 5mL:

1Molarity (M) Tris-HCl, 8.0 50µL

0.5Molarity (M) EDTA 10µL

RNase-free water 4940µL

Aliquot into nulcease-free microfuge tubes. DE Buffer can be stored at Room temperature.

80% volume Ethanol

Combine 8 volumes of ethanol with 2 volumes of nuclease-free water, respectively. Always measure the volumes of both ethanol and water rather than pouring the reagents into a graduated vessel. It is recommended to prepare 15 mL of this at the start of or early on in day 1 and use this throughout day 1 and day 2 until more needs to be prepared on Day 2. *This reagent is only good for 2 days. If it has been more than 36 hrs since it was prepared fresh reagent should be prepared. I.E. If the prep goes into a 3rd day you will need to make more!

PE1 and PE2 primer mixes.

Resuspend PE1 and PE2 primers at 100micromolar (µM) in DE Buffer. Dilute all indexing primers (PE1 and PE2) to 10micromolar (µM) (PE2-N6 should be 100 uM starting concentration). Then mix PE1 primer with its corresponding PE2 primer in a 1:1 ratio. The primer mix of PE1 and PE2 should thus be 5micromolar (µM) for each primer

AMPure XP Beads

Aliquot AMPure XP beads in 2mL nuclease-free tubes. Store at 4°C and bring to Room temperature the day of use. Our current longest test show that AMPure beads are stable at Room temperature for 2 weeks. Always vortex the AMPure XP beads before using them to ensure they are fully resuspended.

2x KAPA HiFi HotStart Readymix

Prepare 300-310 uL aliquots of the readymix. These should then be stored at -20°C

Before starting on Day 1 do the following:

Cool bench-top microcentrifuge to 4°C.
Adjust a heat block to 37°C.
It is generally recommended to plan out which indexes will be used for each sample that will be sequenced together prior to starting. This is not 100% necessary though and can be done during the RT incubation on Day 2 if needed.

Steps

Oligo Removal

This marks the start of Day 1:

Before starting do the following:

Cool bench-top microcentrifuge to 4°C.
Adjust a heat block to 37°C.
It is generally recommended to plan out which indexes will be used for each sample that will be sequenced together prior to starting. This is not 100% necessary though and can be done during the RT incubation on Day 2 if needed.

For each sample pre-chill 1 1.5mL DNA LoBind Tube and 1 Spin-X Column + Tube On ice. Move a 2 mL aliquot of AMPure XP beads to Room temperature to start equilibrating. If a heat

If necessary, thaw samples on ice.

While samples are thawing, visually estimate the volume of each sample. Record this value

For every 70 uL of post-RT material prepare 100 uL of Digest mix as follows:

Component:

Ultrapure RNase/DNase free H₂O 86µL
10X Fast Digest Buffer 9µL
Exonuclease I 5µL
Note
While the samples are thawing mix the H2O and digest buffer. Then add and mix the Exonuclease I to the Digest mix while the sample are spinning in step 4.

Centrifuge tubes at 4°C for 0h 5m 0s at 16000x g,0h 0m 0s to pellet residual HFE (oil).

Adjust the pipette to the estimated sample volume and then block pipette tip with digest mix prior to using it to transfer the sample.

Without drawing up HFE-7500 oil from the bottom of the tube, carefully transfer the sample to a Costar Spin-X column.

Note

Once the sample has been drawn into the pipette tip carefully adjust the pipette volume to the exact sample volume. Record this volume. Then eject the sample onto the column. Eject the sample directly above the filter column. Do not let the sample run down the sides of the filter column.

Repeat steps 7 — 8 for all samples.

10.

Add 100µL of digest mix to the filter column for every 70µL of sample. Recommended to record the volume added.

Example calculation: 70 uL sample / 0.7 = 100 uL of digest to be added.
Note
Do not stick the pipette tip into the sample volume. Pipette the digest mix out directly above the sample.

11.

Centrifuge filter column and collection tube at 4°C for 0h 5m 0s at 16000x g,0h 0m 0s to pass sample through the filter column.

11.1.

During this spin use the previously measured sample volume and digest mix volume to calculate the total volume

12.

Place the samples back On ice. Adjust the pipet tip to the total volume. Block tip with digest mix. Carefully transfer the sample from the collection tube to 1.5 mL DNA LoBind tube.

Note

Once the sample has been drawn into the pipette tip carefully adjust the pipette volume to the exact sample volume. Record this volume. Then eject the sample into the 1.5mL DNA LoBind tube. Eject the sample at the bottom of the tube. Do not let the sample run down the sides of the tube.

13.

Incubate samples at 37°C for 0h 30m 0s.

Note

During this incubation place the 10X Second Strand Synthesis Reaction Buffer (10X SSS buffer) On ice to start thawing.

13.1.

During the incubation calculate the volume of AMPure XP Beads that will be used in the following 0.8x AMPure purification (AMPurification).

Note

Example: (165µL sample volume) x 0.8 = 132µL AMPure XP Beads

14.

Vortex the AMPure bead aliquot to resuspend AMPure beads (5-10 seconds is sufficient).

15.

Add the appropriate volume of AMPure Beads to each sample and mix by pipetting until no mixing lines are observed (use the same tip that was used to add the AMPure beads to mix the sample). DO NOT VORTEX THE SAMPLE.

16.

Incubate each sample/bead mixture at Room temperature for 0h 5m 0s.

17.

Place each sample tube on a magnetic tube rack for 0h 2m 0s at Room temperature or until the beads have fully separated from the supernatant.

Equipment

Value	Label
Magnetic Stand	NAME
Magnetic Stand	TYPE
Thermo Scientific	BRAND
MR02	SKU
Any magnetic rack that fits your tubes will suffice.	SPECIFICATIONS

18.

Carefully aspirate the supernatant without removing/disturbing the bead pellet.

Note

While doing this, slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip.

19.

Wash the Bead pellet with 80% volume ethanol twice at Room temperature.

19.1.

For a single sample: Keeping the tube on the magnet rack gently wash the beads with 400µL of 80% volume ethanol. Rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet.

Note

Pipette the ethanol onto the opposite side of the tube from the beads so as to not greatly disturb the bead pellet. If some beads do not passing from one side of the tube to the other this can sometimes be remedied by taping on the side of the tube where the beads are stuck. If this does not work it is recommended to use the beads that do pass to the other side of the tube to loosen the beads that are stuck by slowly moving the tube around on the magnet such that the beads that were stuck are closest to the magnet.

19.2.

Repeat Step 19.1 for each sample.

19.3.

Again rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet. All tubes should now be in their original orientation.

19.4.

Use a pipette to aspirate and discard the Ethanol. Pipette slowly on the opposite side of the tube from the bead pellet. Again do not disturb the bead pellet with the pipette tip.

19.5.

Repeat steps 19.1 - 19.4 to for the second 80% volume ethanol wash.

20.

After the second ethanol wash quick-spin the sample tubes in a mini-centrifuge for 0h 0m 1s at Room temperature to collect any residual ethanol at the bottom of the tube.

Equipment

Value	Label
Mini-centrifuge	NAME
Centrifuge	TYPE
Fisher	BRAND
S67601B	SKU
Any standard mini centrifuge with adapters for different tube sizes will suffice	SPECIFICATIONS

21.

Place the tubes back on the magnet rack and then use a P200 or smaller pipette to remove any residual ethanol from the bottom of the tube. Do not touch the bead pellet!

22.

Leaving the tubes open on the magnetic tube rack allow each bead pellet to air dry (normally 5-10 min) at Room temperature. While samples are drying get out 1 PCR tube for each sample (do not use strip tubes or plates) and pre-chill on the 96 well aluminum block On ice.

Note

Place a cover over the tubes to prevent dust from falling in them. The pellet will need to dry for at least 5 min. The bead pellet will be a matte black when it is dry and potentially starting to show 1 or 2 cracks. Do not allow the bead pellet to become completely cracked as this will reduce elution efficiency. It is completely normal for some sample to take longer to dry than others (usually larger samples take longer to dry).

23.

Once a sample is dry add 17µL Ultrapure DNase/RNase free H₂O to just that dry sample and place on a normal tube rack at Room temperature.

24.

Gently flick the bottom of the tube to re-suspend the bead pellet in the H₂O.

Note

Avoid flinging the solution onto the top half of the tube. Avoid flinging the solution onto the inside of the lid. DO NOT VORTEX!

25.

Incubate the sample at Room temperature for 0h 5m 0s on a normal tube rack. Proceed to step 26 while waiting.

Note

RNA/DNA will not elute if samples are kept cold during this step.

26.

While the samples are eluting, mix the thawed 10X SSS buffer by pipetting and add 2µL of 10X SSS buffer to each chilled PCR tube.

27.

Spin the sample tubes in a mini-centrifuge for 0h 0m 1s at Room temperature to collect everything at the bottom of the tube.

28.

Place the tube on the magnet rack for about 0h 0m 10s to pellet the beads on the side of the tube.

Note

If not all of the beads pellet against the magnet that is okay. This is caused by either high protein content/debris left over from the encapsulation or a small amount of residual ethanol. The beads can be removed in the following step.

29.

Use a P20 pipette to transfer the supernatant of each sample to its respective chilled PCR tubes and mix by pipetting. If the sample volume is less than 17 uL adjust the volume of the sample up to 17 uL using a P2.

Note

Carrying the magnetic AMPure beads forward should be avoided. Residual AMPure beads can be removed by pipetting the sample out onto the side of the 1.5mL DNA LoBind tube directly above the magnet. Slowly pipette the solution up and down 2-3 times to dislodge any beads stuck to the pipette tip. Allow the beads to pellet against the top of the magnet. Then slowly draw the supernatant back into that same pipette tip while being careful to avoid drawing up any beads that may be present. Throughout this entire process USE A SINGLE PIPETTE TIP PER SAMPLE.It is very important that the total volume added to the tube is 17 uL of sample in H2O going into SSS.

Second Strand Synthesis (SSS)

30.

To each chilled PCR tube add 1 uL of NEBNext Second Strand Synthesis Enzyme mix. Mix the sample by pipetting until few to no mixing lines are observed (10 pipette up and down motions while stirring with the pipet).

31.

Start a PCR machine following the below protocol:

16°C for 2h 30m 0s

65°C for 0h 20m 0s

4°C hold

Lid: 50°C

Do not place the samples on the PCR machine until the PCR block is at 16°C. Once the PCR block temperature is at 16°C the samples should be placed on the incubator to start the SSS reaction.

Note

Proceed to preparing the In Vitro Transcription reaction while the SSS reaction is on-going.

In Vitro Transcription (IVT)

32.

When the SSS reaction has 50 minutes remaining move the contents of the NEB HiScribe T7 High Yield RNA Synthesis Kit to an ice bucket

to start thawing On ice (leave the T7 enzyme at -20°C).

33.

When the SSS reaction has 10-15 minutes remaining prepare enough of the following IVT master mix On ice for each sample using the NEB HiScribe T7 Kigh Yield RNA Synthesis Kit reagents (mix by pipetting):

    Reagent                                                    Volume/Reaction

UltraPure DNase/Rnase-free H2O 10µL

NEB T7 High Yield 10x rxn buffer 8µL

ATP 8µL

CTP 8µL

GTP 8µL

UTP 8µL

NEB T7 High Yield Enzyme Mix 8µL

Pyrophosphatase (NEB M0361S) 2µL

34.

When the SSS reaction has completed, quick-spin the samples in PCR tubes for 0h 0m 2s at Room temperature.

35.

At Room temperature add 60µL of IVT master mix to each sample's PCR tube. Mix by pipetting until no mixing lines are observed.

36.

Start a PCR machine to the follow the below protocol:

37°C for 15h 0m 0s

Lid: 50°C

Do not place the samples on the PCR machine until the PCR block is at 37°C. Once the PCR block temperature is at 37°C the samples should be placed on the incubator to start the SSS reaction.

37.

This marks the start of Day 2:

Prepare an ice bucket (pack the ice). On ice chill the 96 well aluminum block. Prepare a single 1.5 mL DNA LoBind tube for each sample.

38.

Vortex the AMPure bead aliquot to resuspend the AMPure XP beads (5-10 seconds is sufficient for a 2mL aliquot).

39.

Add the 72 uL of AMPure Beads to each sample and mix by pipetting until no mixing lines are observed (use the same tip that was used to add the AMPure beads to mix the sample). DO NOT VORTEX THE SAMPLE.

40.

Incubate each sample/bead mixture at Room temperature for 0h 5m 0s.

41.

Place each sample tube on a magnetic tube rack for 0h 2m 0s at Room temperature or until the beads have fully separated from the supernatant.

Equipment

Value	Label
Magnetic Stand	NAME
Magnetic Stand	TYPE
Thermo Scientific	BRAND
MR02	SKU
Any magnetic rack that fits your tubes will suffice.	SPECIFICATIONS

42.

Carefully aspirate the supernatant without removing/disturbing the bead pellet.

Note

While doing this, slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip.

43.

Wash the Bead pellet with 80% volume ethanol twice at Room temperature.

43.1.

Note

43.2.

Repeat Step 43.1 for each sample.

43.3.

Again rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet. All tubes should now be in their original orientation.

43.4.

Use a pipette to slowly aspirate and discard the Ethanol. Draw the ethanol up from the opposite side of the bead pellet. Do not disturb the bead pellet with the pipette tip.

43.5.

Repeat steps 19.1 - 19.4 to for the second 80% volume ethanol wash.

44.

After the second ethanol wash, quick-spin the sample tubes in a mini-centrifuge for 0h 0m 1s at Room temperature to collect any residual ethanol at the bottom of the tube.

Equipment

Value	Label
Mini-centrifuge	NAME
Centrifuge	TYPE
Fisher	BRAND
S67601B	SKU
Any standard mini centrifuge with adapters for different tube sizes will suffice	SPECIFICATIONS

45.

Place the tubes back on the magnet rack and then use a P200 or smaller pipette to remove any residual ethanol from the bottom of the tube. Do not touch the bead pellet!

46.

Leaving the tubes open on the magnetic tube rack allow each bead pellet to air dry for 0h 5m 0s at Room temperature. While samples are drying get out 1 PCR tube and one 0.5 mL DNA LoBind tube for each sample (do not use strip tubes or plates) and pre-chill On ice. Use the 96 well aluminum block for the PCR tubes.

Note

Place a dust-free cover over the drying tubes to prevent dust from falling into them. All samples should be dry after 5 minutes. On rare occasions a sample may take 6 or 7 minutes to dry.

47.

Once a sample is dry, add 20µL RNA Elution buffer to just that dry sample and place on a normal tube rack at Room temperature.

48.

Gently flick the bottom of the tube to re-suspend the bead pellet in the H₂O.

Note

Avoid flinging the solution onto the top half of the tube. Avoid flinging the solution onto the inside of the lid. DO NOT VORTEX!

49.

Incubate the sample at Room temperature for 0h 5m 0s on a normal tube rack. During this time prepare 1 Qubit tube for each sample, 2 Qubit tubes for standards, and one 2 mL tube for preparing the Qubit reagents.

Note

RNA/DNA will not elute if samples are kept cold during this step.

50.

Spin the sample tubes in a mini-centrifuge for 0h 0m 1s at Room temperature to collect everything at the bottom of the tube.

Note

Spinning for longer than 2 seconds here can result in AMPure beads getting stuck at the bottom of the tube at step 51.

Equipment

Value	Label
Mini-centrifuge	NAME
Centrifuge	TYPE
Fisher	BRAND
S67601B	SKU
Any standard mini centrifuge with adapters for different tube sizes will suffice	SPECIFICATIONS

51.

Place the tube on the magnet rack for about 0h 0m 10s to pellet the beads on the side of the tube.

Note

If not all of the beads pellet against the magnet that is okay. They can be removed in the following step.

52.

Use a P20 pipette to transfer the supernatant (20µL) of each sample to its respective chilled 0.5 mL DNA LoBind tube. Using the same pipette tip transfer 9µL of the sample to it's respective chilled PCR tube. Repeat this process for the remaining samples. The PCR tube is now the sample tube. The 0.5 mL DNA LoBind tube is now the backup.

Note

Carrying the magnetic AMPure beads forward should be avoided. Residual AMPure beads can be removed by pipetting the sample out onto the side of the 1.5mL DNA LoBind tube directly above the magnet. Slowly pipette up and down 2-3 times, and then allow the beads to cluster on the magnet for 2-3 seconds. Then slowly draw the supernatant back into that same pipette tip while being careful to avoid drawing up any beads that may be present.

53.

Use 2 uL from the backup tube to measure the RNA concentration of the sample via Qubit using the Qubit RNA HS assay kit. At this point it is also optional to use 1 uL of the sample for evaluation using a BioAnalyzer RNA Pico Chip Assay.

Note

The Qubit RNA HS reagent from the Qubit RNA HS assay kit is very light sensitive. Only use it with the lights off.

Citation

RNA yields are highly dependent on the types of cells and cell viability in the sample during encapsulation.For mouse/human intestinal epithelium cells:0-20 ng/uL RNA is low.20-60 ng/uL of RNA is normal.60+ ng/uL of RNA is high.If the RNA yield exceeds 85 ng/uL then the sample should be diluted to 80 ng/uL before proceeding to fragmentation.If the Qubit output is "TL" (i.e. Too Low) a library can still sometimes be prepared, but all of the sample (18µL )should be carried forwards (18µL).

Note

If samples will be further processed another day this is a safe pause point. Samples can be stored at -80°C.

Fragmentation

54.

According to 1CellBio: if the samples were run on a BioAnalyzer and the average size <800 bp fragmentation then it is not recommendd to fragment the library, and one can proceed Reverse Transcription using random hexamers.

If samples were thawed from backup tubes transfer 9µL to a PCR tube. Top off any missing volume with RNA Elution Buffer. Otherwise proceed to step 55.

55.

Set up a thermal cycler to follow the below protocol:

70°C hold

lid: 105°C

56.

Prepare the following Fragmentation Stop mix in a DNA LoBind tube and keep at room temperature:

A	B
Component	Volume per Reaction
Ultrapure DNase/RNase-free H2O	9 uL
10x Fragmentation Stop Solution	1 uL
AMPure Beads	16 uL
Total	26 uL

57.

For each sample Pre-chill a single 1.5mL DNA LoBind tube On ice. Also pre-set a P200 to 26 uL. Steps 58-61 should be completed as quickly as possible after this.

58.

Warning: Wait to do this step until the thermal cycler is at 70°C. Add 1 uL of 10X RNA fragmentation reagent to each sample. Mix by pipetting (5 times) and stirring. Immediately place back on chilled 96 well aluminum block.

59.

Once the fragmentation reagent has been added to all samples, place all samples on the pre-heated thermal cycler for 1-2 minutes. Refer to the below table for guidance with regards to fragmentation duration.

A	B
IVT yield ranges (ng/uL)	Example fragmentation durations for intestinal epithelium cell libraries (m:ss)
0-2	0:00 to 0:20
2-10	0:20 to 1:00
10-40	1:00 to 1:30
40-60	1:30 to 1:45
60-80	1:40 to 1:50
80+	1:50 to 2:00

60.

After fragmentation immediately place all samples on the chilled 96 well aluminum block.

61.

Add 26 uL of Fragmentation Stop mix to each sample and transfer that sample to its pre-chilled 1.5 mL DNA LoBind tube.

Note

For minimal sample loss Use a P200 pipette to add 26 uL of Fragmentation Stop mix to a single sample. Using the same pipette tip immediately transfer that sample to the 1.5 mL tube. Mix by pipetting until no mixing lines are observed. Repeat this for all samples (use a new pipette tip for each sample).

62.

Incubate the samples on ice for 0h 5m 0s. At this time place aliquots of the 1x PrimeScript RT buffer, the 100micromolar (µM) TruDrop PE2-N6 primer, and dNTPs On ice to start thawing.

63.

Place each sample tube on a magnetic tube rack for 0h 2m 0s at Room temperature or until the beads have fully separated from the supernatant.

Equipment

Value	Label
Magnetic Stand	NAME
Magnetic Stand	TYPE
Thermo Scientific	BRAND
MR02	SKU
Any magnetic rack that fits your tubes will suffice.	SPECIFICATIONS

64.

Use a P200 pipette to carefully aspirate the supernatant without removing/disturbing the bead pellet.

Note

While doing this, slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip.

65.

Wash the Bead pellet with 80% volume ethanol twice at Room temperature.

65.1.

Note

Pipette the ethanol onto the opposite side of the tube from the beads so as to not greatly disturb the bead pellet. If some beads do not pass from one side of the tube to the other, this can sometimes be remedied by taping on the side of the tube where the beads are stuck. For this AMPurification in particular speed is generally prioritized over passing all beads across the tube so one should proceed to step 65.2. If one would still like to have stuck beads pass across the tube then follow the instructions below.If this does not work it is recommended to use the beads that do pass to the other side of the tube to loosen the beads that are stuck by slowly moving the tube around on the magnet such that the beads that were stuck are closest to the magnet. At this point, slightly moving the tube up and down with some rotation can then sometimes dislodge the beads that are stuck. The tube can then be rotated 180° again to pass all other beads across the tube. If some beads are still stuck after this it is generally recommended to proceed anyways. It is not essential for all beads to pass across the tube though it is recommended.

65.2.

Repeat Step 65.1 for each sample.

65.3.

Again rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet. All tubes should now be in their original orientation.

65.4.

Use a pipette to slowly aspirate and discard the Ethanol for each sample. Draw the ethanol up from the opposite side of the bead pellet. Do not disturb the bead pellet with the pipette tip.

65.5.

Repeat steps 65.1 - 65.4 to for the second 80% volume ethanol wash.

66.

After the second ethanol wash, quick-spin the sample tubes in a mini-centrifuge for 0h 0m 1s at Room temperature to collect any residual ethanol at the bottom of the tube.

Equipment

Value	Label
Mini-centrifuge	NAME
Centrifuge	TYPE
Fisher	BRAND
S67601B	SKU
Any standard mini centrifuge with adapters for different tube sizes will suffice	SPECIFICATIONS

67.

Place the tubes back on the magnet rack and then use a P200 or smaller pipette to remove any residual ethanol from the bottom of the tube. Do not touch the bead pellet!

68.

Leaving the tubes open on the magnetic tube rack allow each bead pellet to air dry for 0h 5m 0s at Room temperature. While samples are drying, do the following:

Set up and start a thermal cycler with the following protocol: 65°C hold
```
      Lid: `105°C` 
```
Pre-chill 1 PCR tube for each sample On ice. Use the 96 well aluminum block for the PCR tubes.
If possible, add 1 uL of dNTP's with 2 uL of TruDrop PE2-N6 primer in the PCR tube (otherwise this can be done later).
Note
Place a dust-free cover over the tubes to prevent dust from falling into them. All samples should be dry after 5 minutes. On rare occasions a sample may take 6 or 7 minutes to dry.Always mix the PE2-N6 primer and dNTP's by pipetting before using it or aliquoting it out. This should be done until no mixing lines are observed.

69.

Once a sample is dry add 8µL RNA Elution buffer to just that dry sample and place on a normal tube rack at Room temperature.

70.

Gently flick the bottom of the tube to re-suspend the bead pellet in the RNA Elution buffer.

Note

Avoid flinging the solution onto the top half of the tube. Avoid flinging the solution onto the inside of the lid. DO NOT VORTEX!

71.

Incubate the sample at Room temperature for 0h 5m 0s on a normal tube rack. During this time perform the following:

finish adding 1µL of dNTPs and 2µL of TruDrop PE2-N6 primer to the pre-staged PCR tubes.
Start preparing the following RT mastermix in a DNA LoBind Tube (only add the H2O and RT buffer):

A	B
Reagent	Volume Per Reaction
Ultrapure DNase/RNase-free H2O	2 μL
5x PrimeScript RT buffer	4 μL
RNase OUT (40 U/μL)	2 μL
PrimeScript RT enzyme (200 U/μL)	1 μL
total	9 μL

Note

RNA/DNA will not elute if samples are kept cold during this step.Always mix the PE2-N6 primer, dNTP's, and RT buffer by pipetting before using it or aliquoting it out. This should be done until no mixing lines are observed.

72.

Spin the sample tubes in a mini-centrifuge for 0h 0m 1s at Room temperature to collect everything at the bottom of the tube.

Note

Spinning for longer than 2 seconds here can result in AMPure beads getting stuck at the bottom of the tube at step 51.

Equipment

Value	Label
Mini-centrifuge	NAME
Centrifuge	TYPE
Fisher	BRAND
S67601B	SKU
Any standard mini centrifuge with adapters for different tube sizes will suffice	SPECIFICATIONS

73.

Place the tube on the magnet rack for about 0h 0m 10s to pellet the beads on the side of the tube.

Note

If not all of the beads pellet against the magnet that is okay. They can be removed in the following step.

74.

Use a P20 pipette to transfer the supernatant (8µL) of each sample to its respective chilled PCR tube. Pipette up and down (5x) to mix the sample with the primer and dNTPs.

Note

Carrying the magnetic AMPure beads forward should be avoided. Residual AMPure beads can be removed by pipetting the sample out onto the side of the 1.5mL DNA LoBind tube directly above the magnet. Slowly pipette up and down 2-3 times, and then allow the beads to cluster on the magnet for 2-3 seconds. Then slowly draw the supernatant back into that same pipette tip while being careful to avoid drawing up any beads that may be present.

2nd Reverse Transcription

75.

If it has not been done yet, start preparing the following RT mastermix in a DNA LoBind Tube (only add the H2O and RT buffer):

A	B
Reagent	Volume Per Reaction
Ultrapure DNase/RNase-free H2O	2 μL
5x PrimeScript RT buffer	4 μL
RNase OUT (40 U/μL)	2 μL
PrimeScript RT enzyme (200 U/μL)	1 μL
total	9 μL

Note

Always mix the RT buffer by pipetting before using it or aliquoting it out. This should be done until no mixing lines are observed.

76.

Incubate all samples at 65 for 0h 3m 0s on the thermal cycler set up in step 68. During this time finish making the RT master mix (step 75, add the RNase OUT and PrimScript RT enzyme). Mix by pipetting.

77.

Remove all samples form the thermal cycler and cool on ice (use the chilled 96-well block for about 5-10 seconds) before adding 9 uL of RT master mix to each sample. Using the same pipette tip that was used to add the RT mix, mix the sample by pipetting.

78.

Start a thermal cycler following the below protocol:

30°C for 0h 10m 0s

42°C for 1h 0m 0s

70°C for 0h 15m 0s

4°C hold

lid: 105°C

Place the samples on the thermal cycler heat-block when it reaches 30°C. During this time prepare two 1.5 mL DNA LoBind tubes for each sample. Pre-chill one of the two On ice. Keep the other one at room temperature. If more 80% volume ethanol will be needed now is a great time to prepare it.

Note

During this time if the sample indices have not already been chosen for each sample that should be done here. The PCR reactions after the RT cannot be completed without knowing which samples will have which index pairs. For assistance in picking which indices can be used together it is recommended to follow the indexing guidelines in the Illumina document "index-adapters-pooling-guide-1000000041074-08" (specifically the section: "IDT for Illumina-TruSeq UD Indexes").Further indexing guidelines can be found in the Sequencing section (step 130) of this protocol.

79.

Place all samples on the chilled 96 well block On ice. Add 30µL of Ultrapure DNase/RNase-free H₂O to each sample.

80.

Use a P200 pipette to add 35µL of Ampure XP beads to a single sample. Using the same pipette tip, transfer the sample to a room temperature 1.5 mL DNA LoBind Tube and pipette up and down until no mixing lines are observed. DO NOT VORTEX.

80.1.

Repeat step 93 for each sample.

81.

Incubate each sample/bead mixture at Room temperature for 0h 5m 0s.

During this time do the following:

If needed, prepare more 80% volume ethanol. Otherwise:
On ice start thawing the TruDrop PE1/PE2 indexed 5micromolar (µM) primer mixes that will be used for each sample.
Finish preparing a 1.5 mL DNA LoBind tube for each sample and pre-chill on ice.

82.

Place each sample tube on a magnetic tube rack for 0h 2m 0s at Room temperature or until the beads have fully separated from the supernatant.

During this time do the following:

On ice start thawing the TruDrop PE1/PE2 indexed 5micromolar (µM) primer mixes that will be used for each sample.
Note
At the end of the 2 minutes the pellet can still look slightly diffuse. This is normal. Proceed to the next step anyways. The post-2^nd-RT AMPurification can be very finicky. The diffuse nature of the beads is due to either the high glycerol concentration from the RT, or the large amount of protein from the RT, or both.

Equipment

Value	Label
Magnetic Stand	NAME
Magnetic Stand	TYPE
Thermo Scientific	BRAND
MR02	SKU
Any magnetic rack that fits your tubes will suffice.	SPECIFICATIONS

83.

Carefully aspirate the supernatant without removing/disturbing the bead pellet.

Note

While doing this, use a P200 pipette to slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip. Do not aspirate any beads! If beads are removed by accident they can be recovered as follows:Hold the magnet rack such that the side of the tube at the top of the magnet is horizontal.Slowly pipette the entire solution out onto the top of the magnet making sure the supernatant does not contact the bead pellet. Slowly draw the supernatant up into the pipette, leaving behind any small amount of beads pulled out of solution on the side of the tube. If a 1-2 uL drop of supernatant is left behind that will not be a major problem.

84.

Wash the Bead pellet with 80% volume ethanol twice at Room temperature.

84.1.

Note

Pipette the ethanol onto the opposite side of the tube from the beads so as to not greatly disturb the bead pellet. Then, rotate the tube. If some beads do not passing from one side of the tube to the other this can sometimes be remedied by taping on the side of the tube where the beads are stuck. If this does not work it is recommended to use the beads that do pass to the other side of the tube to loosen the beads that are stuck by slowly moving the tube around on the magnet such that the beads that were stuck are closest to the magnet. At this point, slightly moving the tube up and down with some rotation can then sometimes dislodge the beads that are stuck. The tube can then be rotated 180° again to pass all other beads across the tube. If some beads are still stuck after this it is generally recommended to proceed anyways. It is not essential for all beads to pass across the tube though it is recommended.During the first ethanol wash of the AMPurification after 2nd RT it is very normal for all of the beads to not pass across the tube when rotated on the magnet rack. The best way to avoid this is to rotate the tube ASAP after adding the first ethanol wash. It may take more tapping/moving/rotating the tube than normal to pass all beads across.

84.2.

Repeat Step 97.1 for each sample.

84.3.

Again rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet. All tubes should now be in their original orientation.

84.4.

Use a pipette to slowly aspirate and discard the Ethanol. Draw the ethanol up from the opposite side of the bead pellet. Do not disturb the bead pellet with the pipette tip.

84.5.

Repeat steps 97.1 - 97.4 to for the second 80% volume ethanol wash.

85.

After the second ethanol wash, quick-spin the sample tubes in a mini-centrifuge for 0h 0m 1s at Room temperature to collect any residual ethanol at the bottom of the tube.

Equipment

Value	Label
Mini-centrifuge	NAME
Centrifuge	TYPE
Fisher	BRAND
S67601B	SKU
Any standard mini centrifuge with adapters for different tube sizes will suffice	SPECIFICATIONS

86.

Place the tubes back on the magnet rack and then use a P200 or smaller pipette to remove any residual ethanol from the bottom of the tube. Do not touch the bead pellet!

87.

Leaving the tubes open on the magnetic tube rack, allow each bead pellet to air dry for 0h 2m 0s at Room temperature. If a single 1.5 mL DNA LoBind tube has not yet been pre-chilled for each sample, prepare those now.

Note

Place a dust-free cover over the drying tubes to prevent dust from falling into them. All samples should be dry after 2—2:30 minutes. At this step, it is generally recommended to just elute all samples here after 2 minutes. Allowing samples to over-dry here will result in the pellet becoming cracked very quickly, and them not being able to be fully re-suspended during elution.

88.

Once a sample is dry, add 40µL DNA Elution buffer to just that dry sample and place on a normal tube rack at Room temperature.

89.

Gently flick the bottom of the tube to re-suspend the bead pellet in the DNA Elution buffer.

Note

Avoid flinging the solution onto the top half of the tube. Avoid flinging the solution onto the inside of the lid. DO NOT VORTEX!

90.

Incubate each sample/bead mixture at Room temperature for 0h 5m 0s.

While samples are eluting:

Get out 1 PCR tube for each sample (do not use strip tubes or plates) and pre-chill On ice. Use the 96 well aluminum block for the PCR tubes.
Start thawing a 300 uL aliquot of 2x KAPA HiFi HotStart On ice.

91.

Spin the sample tubes in a mini-centrifuge for 0h 0m 1s at Room temperature to collect everything at the bottom of the tube.

Note

Spinning for longer than 2 seconds here can result in AMPure beads getting stuck at the bottom of the tube at step 51.

Equipment

Value	Label
Mini-centrifuge	NAME
Centrifuge	TYPE
Fisher	BRAND
S67601B	SKU
Any standard mini centrifuge with adapters for different tube sizes will suffice	SPECIFICATIONS

92.

Place each sample tube on a magnetic tube rack for 0h 0m 15s at Room temperature, or until the beads have fully separated from the supernatant.

Note

For the RT Ampurification it is normal to have a thin film of beads that remain and continue to coat the bottom of the tube when placed against the magnet. As long as a distinct small pellet of beads is observed close to the magnet it is okay to proceed to step 106.

93.

Use a P20 pipette to transfer the supernatant (40µL) of each sample to its respective chilled 1.5 mL DNA LoBind tube. Using the same pipette tip transfer 20µL of the sample to it's respective chilled PCR tube. Repeat this process for the remaining samples. The PCR tube is now the sample tube. The 1.5 mL DNA LoBind tube is now the pre-PCR backup.

Note

Carrying the magnetic AMPure beads forward should be avoided. Residual AMPure beads can be removed by pipetting the sample out onto the side of the 1.5mL DNA LoBind tube directly above the magnet. Slowly pipette up and down 2-3 times, and then allow the beads to cluster on the magnet for 2-3 seconds. Then slowly draw the supernatant back into that same pipette tip while being careful to avoid drawing up any beads that may be present.

Note

This can be a pause point. It is highly recommended that all samples proceed with library preparation to finish preparing the final library. However, if necessary the tubes at this stage can be frozen at -80°C until the library preparation can continue.

Diagnostic qPCR

94.

Mix the thawed 2x KAPA and all of the TruDrop primer mixes to be used by pipetting until no mixing lines are observed or at least by pipetting up and down 5x. Next, place a 96 well PCR Plate into a pre-frozen (-20°C) PCR-Cooler rack.

95.

Prepare the following qPCR mixture on ice:

A	B
Reagent	Volume per reaction
Ultrapure DNase/RNase-free H2O	13 μL
2x KAPA HiFi HotStart PCR Mix	20 μL
20x Eva Green Dye	2 μL
Total	35 μL

96.

Decide and record which sample will be PCR'ed in which sample well. It is recommended to space your samples so that none are located on the border of the plate. Example: We always use column 4 starting at row B and ending at row G. Add 35 uL of qPCR mixture to each well that will contain a sample.

97.

Add 4 uL of the TruDrop PE1/PE2 primer mixture that corresponds to each sample to the well of the PCR plate where that sample will be located.

98.

Add 1 uL of a single sample to it's corresponding well in the PCR plate. Make sure to pipette out into the liquid in each well and not onto the side of the plate.

98.1.

Repeat step 111 for each sample

99.

Seal the plate with a MicroSeal (BioRad MSB-100). Use the smooth side of the paper protector to press down around the edges of the wells containing the samples and around the edges of the plate.

100.

Spin the qPCr Plate at 600rpm,4°C to collect everything at the bottom of the well(s) and remove any bubbles.

101.

Set up a qPCR machine with the following protocol, load the plate in the machine and start the PCR machine:

98°C for 0h 2m 0s
98°C for 0h 0m 20s
55°C for 0h 0m 30s
72°C for 0h 0m 40s
Go to 2) once (a total of two cycles)
98°C for 0h 0m 20s
65°C for 0h 0m 30s
72°C for 0h 0m 40s with fluorescent read
Go to 6) 23 times (a total of 24 cycles)

Lid: 105°C

Volume: 40µL

Note

This PCR normally takes about 1 hour 25 minutes. While the reaction is going the remaining sample (19.5 uL) PCR tubes, primer tubes, and KAPA should be placed on the chilled 96 well aluminum block and then stored in a clean 4oC fridge during the course of the reaction. This is also a good point to replenish the ice in the ice bucket.

102.

Set the qPCR threshold within the exponential phase of amplification, but closer to where the signal starts to emerge from the noise to avoid the possibility of over amplification. We normally wil adjust to about 5000 RFU.

103.

Correct the resulting Ct value to account for differences in the amount of input material between the qPCR and library PCR. The library input is 19 times higher, and assuming a 100% PCR efficiency, the difference corresponds to log2 19 = 4.25 cycles. For example if the number of cycles determined by qPCR is 2 + 10.27, the required number of PCR cycles for library is 2 + (10.27 — 4.25) = 2 + 6.02 . This would then be rounded to 2 + 6 cycles. The rules we use for rounding here is that any number with a decimal value of 0.4 or more is rounded up to the nearest integer, while any number with a decimal value less that 0.4 is rounded down to the nearest integer. Example: 6.38 would be rounded down to 6 cycles, but 6.42 is rounded up to 7 cycles. Record this rounded value as the 2 + N, where N is the rounded adjusted number of cycles.

Citation

Expected result is normally 4-7 cycles depending on the sample.

PCR Amplification

104.

Move the 96 well aluminum block with the samples, primer mixes, and KAPA back to the ice bucket.

105.

Prepare the following PCR mixture On ice:

A	B
Reagent	Volume per reaction
Ultrapure DNase/RNase-free H2O	1 uL
2x Kapa HiFi HotStart PCR mix	25 uL
Total	26 uL

106.

To each sample add 5 uL of TruDrop PE1/PE2 primer mix to the corresponding sample tube.

107.

To each sample add 26 uL of PCR mix to the corresponding sample tube. Mix the sample by pipetting until no mixing lines are observed.

108.

Set up and start a PCR machine to follow the below protocol (wait to place the samples on the machine until the heat block is 95°C ):

98°C for 0h 2m 0s
98°C for 0h 0m 20s
55°C for 0h 0m 30s
72°C for 0h 0m 40s
Go to 2) once (a total of two cycles)
98°C for 0h 0m 20s
65°C for 0h 0m 30s
72°C for 0h 0m 40s with fluorescent read
Go to 6) N-1 times (a total of N adjusted cycles).
72°C for 0h 5m 0s
hold 4°C

Lid: 105°C

Volume: 50µLTo the sample

The value for N was determined as a part of step 116. If working with multiple libraries that require different numbers of cycles, set the thermal cycler to perform the highest number of cycles required, and manually remove individual libraries from the thermal cycler, when they reach the desired cycle number. For example, if library X requires 8 cycles, at the end of the 72°C step of cycle 8 pause the cycler and transfer library X to an ice bucket. Then, resume the PCR program. Once all the samples have undergone the required number of PCR cycles, transfer all tubes back to the thermal cycler for the final step, a 5 min incubation at 72°C.

During the final 5 min extension at 72oC prepare 3 DNA LoBind tubes for each sample. 2 tubes will be kept at Room temperature. The final tube should be chilled On ice. To one of each sample's room temperature LoBind tubes add 20 uL of AMPure beads (part of this can be done later).

109.

When the PCR is finished transfer all samples to the chilled 96-well aluminum block On ice. Add 50µL of DNA Elution buffer to the PCR product.

110.

To a single sample add 60 uL of AMPurebeads. Using the same pipette tip transfer the sample + bead mixture to its empty DNA LoBind tube. Mix by pipetting until no mixing lines are observed. DO NOT VORTEX.

Note

This is the start of a double sided AMPurification. The lower bound is 0.6x (step 110). The upper bound is 0.8x (step 113).

110.1.

Do step 123 for all other samples.

111.

Incubate each sample/bead mixture at Room temperature for 0h 5m 0s.

During this time do the following:

If needed, prepare more 80% volume ethanol.
Finish adding 20 uL of AMPure beads to the 2nd set of room temperature tubes that will be used.

112.

Place each sample tube on a magnetic tube rack for 0h 2m 0s at Room temperature . It is important that samples are on the magnet rack for the full 2 minutes.

Equipment

Value	Label
Magnetic Stand	NAME
Magnetic Stand	TYPE
Thermo Scientific	BRAND
MR02	SKU
Any magnetic rack that fits your tubes will suffice.	SPECIFICATIONS

113.

The finished library is what is in the supernatant. For a single sample, carefully transfer the supernatant to the second room temperature 1.5 mL DNA LoBind tube containing 20 uL of AMPure beads. Do not carry forward any beads. Using the same pipette tip, mix the sample by pipetting until no mixing lines are observed. The second tube is now the sample tube.

Note

While doing this, use a P200 pipette to slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip. Do not aspirate any beads! If beads are removed by accident they can be removed as follows:Close alltubes and hold the magnet rack such that the side of the tube at the top of the magnet is horizontal.Slowly pipette the entire solution out onto the top of the magnet making sure the supernatant does not contact the bead pellet. Slowly draw the supernatant up into the pipette, leaving behind any small amount of beads pulled out of solution on the side of the tube. If a 1-2 uL drop of supernatant is left behind that will not be a major problem.

113.1.

Repeat step 126 for all remaining samples.

114.

Incubate each sample/bead mixture at Room temperature for 0h 5m 0s on a normal tube rack.

115.

Place each sample tube on a magnetic tube rack for 0h 2m 0s at Room temperature . It is important that samples are on the magnet rack for the full 2 minutes.

Equipment

Value	Label
Magnetic Stand	NAME
Magnetic Stand	TYPE
Thermo Scientific	BRAND
MR02	SKU
Any magnetic rack that fits your tubes will suffice.	SPECIFICATIONS

116.

The sample is now what is on the beads. Carefully aspirate the supernatant without removing/disturbing the bead pellet.

Note

117.

Wash the Bead pellet with 80% volume ethanol twice at Room temperature.

117.1.

Note

117.2.

Repeat Step 117.1 for each sample.

117.3.

Again rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet. All tubes should now be in their original orientation.

117.4.

Use a pipette to slowly aspirate and discard the Ethanol. Draw the ethanol up from the opposite side of the bead pellet. Do not disturb the bead pellet with the pipette tip.

117.5.

Repeat steps 117.1 - 117.4 to for the second 80% volume ethanol wash.

118.

After the second ethanol wash, quick-spin the sample tubes in a mini-centrifuge for 0h 0m 1s at Room temperature to collect any residual ethanol at the bottom of the tube.

Equipment

Value	Label
Mini-centrifuge	NAME
Centrifuge	TYPE
Fisher	BRAND
S67601B	SKU
Any standard mini centrifuge with adapters for different tube sizes will suffice	SPECIFICATIONS

119.

Place the tubes back on the magnet rack and then use a P200 or smaller pipette to remove any residual ethanol from the bottom of the tube. Do not touch the bead pellet!

120.

Leaving the tubes open on the magnetic tube rack allow each bead pellet to air dry for 0h 2m 0s at Room temperature. If a single 1.5 mL DNA LoBind tube has not yet been pre-chilled for each sample, prepare those now.

Note

Place a dust-free cover over the drying tubes to prevent dust from falling into them. All samples should be dry after 2—2:30 minutes.

121.

Once a sample is dry, add 20µL DNA Elution buffer to just that dry sample and place on a normal tube rack at Room temperature.

122.

Gently flick the bottom of the tube to re-suspend the bead pellet in the DNA Elution Buffer.

Note

Avoid flinging the solution onto the top half of the tube. Avoid flinging the solution onto the inside of the lid. DO NOT VORTEX!

123.

Incubate each sample/bead mixture at Room temperature for 0h 5m 0s.

124.

Spin the sample tubes in a mini-centrifuge for 0h 0m 1s at Room temperature to collect everything at the bottom of the tube.

Note

Spinning for longer than 2 seconds here can result in AMPure beads getting stuck at the bottom of the tube at step 51.

Equipment

Value	Label
Mini-centrifuge	NAME
Centrifuge	TYPE
Fisher	BRAND
S67601B	SKU
Any standard mini centrifuge with adapters for different tube sizes will suffice	SPECIFICATIONS

125.

Place each sample tube on a magnetic tube rack for 0h 0m 15s at Room temperature, or until the beads have fully separated from the supernatant.

Note

126.

Use a P20 pipette to transfer the supernatant (20µL) of each sample to its respective chilled 1.5 mL DNA LoBind tube. This is the final library.

Note

Carrying the magnetic AMPure beads forward should be avoided. Residual AMPure beads can be removed by pipetting the sample out onto the side of the 1.5mL DNA LoBind tube directly above the magnet. Slowly pipette up and down 2-3 times, and then allow the beads to cluster on the magnet for 2-3 seconds. Then slowly draw the supernatant back into that same pipette tip while being careful to avoid drawing up any beads that may be present.

127.

Use 2 uL from the sample to measure the DNA concentration of the sample via Qubit using the Qubit DNA HS assay kit.

Citation

Sample Concentration should be between 1-3 ng/uL.

128.

Samples should be stored at -80°C until they can be sequenced.

Sequencing

129.

Quantify samples via BioAnalyzer and qPCR prior to sequencing.

130.

If sequencing TruDrop libraries in pools alongside higher percentages of other Illumina compatible libraries (70% of the total pool or more is from other Illumina compatible libraries), then one can use each pair of indexes (one i7, and one i5) once per sequencing lane per sequencing run. There are no other limitations on sequencing index usage, provided that a library can be made from the index pair (do not use pair UDI0009). Example: UDI0025 could be used with UDI0001. So long as the indices aren’t repeated across multiple libraries in a single sequencing pool. This option is recommended so as to not run out of certain primer pair before others.

Disclaimer: Our operating range is 70% at the lowest for the above cases, but I suspect that 50% may also work if necessary. If you are unsure, it is recommended that you consult with your sequencing core/provider on which index pairs to use. If consultation is not possible, you can always follow the criteria for library pooling with 17-24 TruDrop libraries, in that order, and it should work every time. The downside to this option is that certain index primers will be expended at a significantly higher rate resulting in varying turnover rates for index pairs.

If Sequencing 1-8 TruDrop libraries alongside lower percentages of other illumina compatible libraries (70% of the total pool or less is from other illumina compatible libraries) then follow the bellow pooling guidelines:

Use UDI0001, then UDI0002, then UDI0003, then UDI0004, then UDI0005, then UDI0006, then UDI0007, then UDI0008,

Use UDI0017, then UDI0018, then UDI0019, then UDI0020, then UDI0021, then UDI0022, then UDI0023, then UDI0024,

If Sequencing 9-16 TruDrop libraries alongside lower percentages of other illumina compatible libraries (70% of the total pool or less is from other illumina compatible libraries) then follow the bellow pooling guidelines:

If Sequencing 17-24 TruDrop libraries alongside lower percentages of other illumina compatible libraries (70% of the total pool or less is from other illumina compatible libraries) then follow the bellow pooling guidelines:

Use UDI0001, then UDI0002, then UDI0003, then UDI0004, then UDI0005, then UDI0006, then UDI0007, then UDI0008, then UDI0017, then UDI0018, then UDI0019, then UDI0020, then UDI0021, then UDI0022, then UDI0023, then UDI0024, then UDI0010, then UDI0011, then UDI0012, then UDI0013, then UDI0014, then UDI0015, then UDI0016, then UDI0025

Use UDI0017, then UDI0018, then UDI0019, then UDI0020, then UDI0021, then UDI0022, then UDI0023, then UDI0024, then UDI0001, then UDI0002, then UDI0003, then UDI0004, then UDI0005, then UDI0006, then UDI0007, then UDI0008, then UDI0010, then UDI0011, then UDI0012, then UDI0013, then UDI0014, then UDI0015, then UDI0016, then UDI0025

131.

At this point the libraries should be treated like normal TruSeq libraries. Use normal TruSeq sequencing primers.

Libraries have been succesfully sequenced on NovaSeq with the following settings:

Read type: Paired- End

Read 1: 50 cycles or more (Cel Barcodes + UMI)

Read 2: 50 cycles or more (we normally use at least 100 cycles for transcript)

i7 index read: 8 cycles

i5 index read: 8 cycles

InDrop Library Preparation - TruDrop (modified V2) Detailed

Abstract

Before start

Steps

Oligo Removal

Second Strand Synthesis (SSS)

In Vitro Transcription (IVT)

Fragmentation

2nd Reverse Transcription

Diagnostic qPCR

PCR Amplification

Sequencing

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