Barcoded and targeted cDNA library preparation for Oxford Nanopore Technologies sequencing

This protocol is adapted from that provided with NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (NEB, E6420S). Input and component values have been modified from the original kit protocol.

Primer annealing for first strand synthesis

Prepare in a PCR tube on ice. A reaction mix of up to 7µL of total RNA (75ng ) is added to 1µL 2millimolar (mM) TSO-ONT primer, 1µL 10millimolar (mM) dNTPs (NEB) and made up to 9µL with nuclease-free H₂O. Gently invert a few times and spin briefly.

Incubate the mixture in a thermocycler for 0h 5m 0s at 70°C with the heated lid at 105°C , then snap cool on ice for 0h 1m 0s .

Reverse Transcription (RT) and Template Switching

Meanwhile, prepare the reverse transcription mix which consists of 2.5µL NEBNext Single Cell RT buffer (lilac); 0.5µL NEBNext Template Switching Oligo (lilac); 1µL NEBNext Single Cell RT enzyme mix (lilac) and 1.5µL nuclease-free water. Mix by gentle inversion and spin briefly. Add 5.5µL of reverse transcription mix to each sample. Gently invert a few times and spin briefly.

Place the mix in a thermocycler (heated lid at 105°C ):

42°C for 1h 30m 0s

70°C for 0h 10m 0s

hold at 4°C .

cDNA amplification by PCR

Prepare the cDNA amplification mix which consists of 25µL NEBNext Single

Cell cDNA PCR Master Mix (orange);1µL NEBNext Single Cell cDNA PCR Primer (orange);0.25µL NEBNext Cell Lysis Buffer (10X) (white) and13.75µL nuclease-free water. Mix by gentle inversion and spin briefly. Add40µL of cDNA amplification mix to each sample. Gently invert a few times and spin briefly.

PCR cycling conditions:

Heated lid set to 105°C

Initial denaturation:

98°C for 0h 0m 45s

For 14 cycles:

98°C for 0h 0m 10s

65°C for 0h 12m 30s ,

72°C for 0h 3m 0s ,

Final extension:

72°C for 0h 5m 0s

hold at 4°C

Cleanup of Amplified cDNA

Bring Promega Pronex beads to Room temperature for 0h 30m 0s prior to use.

Spin tubes down briefly. 0.85X Promega Pronex beads are added. Gently invert a few times and spin briefly. Incubate at Room temperature on Hula mixer for 0h 5m 0s . Briefly spin tubes and place on magnet. Leave beads to settle for 0h 5m 0s . Perform 2x 80% EtOH washes. Amplified cDNA is eluted in 27µL 1X TE.

Dilute sample 1:5 and run an Agilent HS D5000 screentape

We performed an adapted version of the 'PacBio cDNA Capture Using IDT xGen Lockdown Probes' protocol (Other Documentation - PacBio) and xGen Lockdown Panel.

Barcoded cDNA is equimolar pooled for a total of 1µg cDNA per capture reaction. Prepare in a 0.2mL PCR tube.

1µL of TSO blocker and 1µL of polyT blocker (poly(dT)₃₀) oligonucleotides (both at 1millimolar (mM) ) were added to the pooled cDNA.

Dry the mixture using a DNA vacuum concentrator (set temperature to 45°C ). Do not overdry.

Add IDT 2X hybridisation buffer (8.5µL ), hybridisation buffer enhancer (2.7µL ) and nuclease free water (1.8µL ) to the dried-down sample. Replace the lid (do not cover the hole with tape). Mix the reaction by gently tapping the tube, followed by a quick spin.

Place the mixture on a 95°C thermocycler for 0h 10m 0s to denature the cDNA.

After allowing the mixture to cool briefly for 0h 2m 0s at Room temperature. 4µL of xGen Lockdown Panel is added for a total volume of 17µL. Probes should never be added while at 95°C .

After 0h 5m 0s at Room temperature, briefly spin the tubes and incubate in a thermocycler at 65°C overnight (lid temperature 100°C).

Prepare the wash buffers and Dynabeads M-270 Streptavidin beads provided in the xGen Lockdown Hybridisation and Wash kit and use as per instructions below. The xGen Lockdown Hybridisation and Wash kit is optimal for 3 months at -20°C and poor yields may occur if the kit has expired.

Prepare wash buffers

A	B	C	D	E	F
Wash buffer I	10X	40 µL	360 µL	400 µL	1X
Wash buffer II	10X	20 µL	180 µL	200 µL	1X
Wash buffer III	10X	20 µL	180 µL	200 µL	1X
Stringent wash buffer	10X	50 µL	450 µL	500 µL	1X
Bead wash buffer	2X	250 µL	250 µL	500 µL	1X

These volumes are for a single sample - scale up for multiple samples.

Preheat the following wash buffers to 65°C in a heat block or water bath for at least 0h 15m 0s :

200µL of 1X wash buffer I

400µL of 1X stringent wash buffer

Other reagents can be kept at Room temperature

Prepare the capture beads

1. Allow the Dynabeads M-270 Streptavidin to warm to room temperature for 0h 30m 0s prior to use.
2. Mix the beads thoroughly by vortexing for 0h 0m 15s
3. For a single sample, aliquot 100µL beads into a 1.5mL LoBind tube. Scale up volume for multiple samples.
4. Place the LoBind tube in a magnetic rack. When the supernatant is clear, remove and discard the supernatant being careful not to disturb the beads. Any remaining traces of liquid will be removed with subsequent wash steps. Give it 0h 5m 0s to settle. The Dynabeads are "filmy" and slow to collect to the side of the tube.
5. While the LoBind tube is in the magnetic rack, add 200µL of 1X bead wash buffer. For multiple samples, wash with 200µL x X samples.
6. Remove the tube from the magnetic rack and vortex until the beads are in solution.
7. Quickly spin and place the LoBind tube back in the magnetic rack to collect the beads to the side of the tube. Once clear, remove and discard the liquid.
8. Repeat steps 5-7 for a total of two washes.
9. Resuspend by vortexing the beads in 100µL of 1X bead wash buffer. For multiple samples, scale up accordingly.
10. Place the tube in the magnetic rack to collect beads to the side of the tube. Once clear, remove and discard the supernatant.
11. The washed beads are now ready to bind the captured DNA. Proceed immediately to the next step. Do not allow the capture beads to dry. Small amounts of residual bead wash buffer will not interfere with binding of DNA to the capture beads.

Bind cDNA to the capture beads

Transfer the 17µL hybridised probe/sample mixture to the washed capture beads.

Mix by tapping the tube until the sample is homogenous.

Incubate in a heat block set to 65°C for 0h 45m 0s or transfer the mix to a PCR tube and incubate in a thermocycler (heated lid set to 75°C ). Hand mix periodically by gently tapping the tube to keep the beads in suspension, every 0h 10m 0s .

Wash the captured cDNA

1. The 1X wash buffer I and 1X stringent wash buffer should have been pre-heated to 65°C .
2. After the above incubation, remove the tube from the heat block and add 100µL pre-heated 1X wash buffer I.
3. Mix thoroughly by tapping the tube until the sample is homogenous.
4. If using a PCR tube, transfer the sample to a 1.5mL LoBind tube (careful of bubbles).
5. Place the tube in a magnetic rack to collect the beads to the side of the tube. Remove and discard the liquid once clear.
6. Remove the tube from the magnetic rack and add 200µL of 1X stringent wash buffer heated to 65°C . Mix by tapping the tube until the sample is homogenous. Work quickly so that the temperature does not drop.
7. Incubate at 65°C for 0h 5m 0s .
8. Repeat steps 5-7 for a total of two washes using 1X stringent wash buffer heated to 65°C .
9. Place the tubes in the magnetic rack to collect the beads to the side of the tube. Remove and discard the liquid once clear.
10. Add 200µL of Room temperature 1X wash buffer I. Hand mix by gently tapping the tube, followed by a quick spin.
11. Place the tube in the magnetic rack to collect the beads to the side of the tube. Remove and discard the liquid once clear. It will clear quickly but allow 0h 1m 0s .
12. Add 200µL of Room temperature 1X wash buffer II. Hand mix by gently tapping the tube, followed by a quick spin.
13. Place the tube in the magnetic rack to collect the beads to the side of the tube. Remove and discard the liquid once clear. Allow 0h 5m 0s .
14. Add 200µL of Room temperature 1X wash buffer III. Hand mix by gently tapping the tube. Quick spin.
15. Place the tube in the magnetic rack to collect the beads to the side of the tube. Remove and discard the liquid once clear. Allow 0h 5m 0s .
16. Remove the tubes from the magnetic rack and add 50µL of 1x TE.
17. Store the beads plus captured samples at -20°C or proceed to the next step. It is not necessary to separate the beads from the eluted DNA.

Amplification of captured DNA sample

A	B
Water	104.5 µL
10x LA PCR buffer	20 µL
2.5 mM dNTPs	16 µL
12 uM TSO amp primer	8.3 µL
Takara LA Taq DNA polymerase	1.2 µL
Captured library	50 µL
Total	200 µL

PCR reaction mix

Split the PCR mix into two tubes, 100µL each.

Amplify using the following PCR protocol:

A	B	C
1	95 °C	2 mins
2	95 °C	20 s
3	68 °C	10 mins
4	Repeat steps 2-3 for a total of 11 cycles
5	72 °C	10 mins
6	4 °C	hold

After amplification, pool the 100µL reactions.

Post amplification clean up

Bring AMPure beads to Room temperature for 0h 30m 0s prior to use.

Spin tubes down briefly. 1X AMPure beads are added. Gently invert a few times and spin briefly. Incubate at Room temperature for 0h 10m 0s . Briefly spin tubes and place on magnet. Leave beads to settle for 0h 5m 0s . Perform 2x 80% EtOH washes. Amplified cDNA is eluted in 27µL 1X TE.

Quantification was determined using the Qubit DNA High sensitivity assay

(Invitrogen, UK) (1:5 dilution) and HS D5000 screentape (Agilent, UK) (1:5 dilution).

Library preparation was performed using ONT’s ligation kit (SQK-LSK114). Follow supplier protocol, which also includes details for sequencing. Recommended library input is 100-200 fmol.