Reduced Representation Bisulfite Sequencing (RRBS) with NEB Reagents

Noah Noah Snyder-Mackler

Published: 2023-05-09 DOI: 10.17504/protocols.io.e6nvwkxb9vmk/v1

Abstract

This protocol is for generating Reduced Representation Bisulfite Sequencing (RRBS) libraries. We recommend using 200ng input, but the protocol has worked with inputs as low as 50ng.

We recommend using a pippin prep to remove small library fragments prior to sequencing.

We see the best results when we size select between 180bp-2000bp and sequence using single end (at least 50 base) reads on an Illumina NovaSeq.

NEB 10bp dual index sequences

Before start

Suggested schedule:

Day 1

Morning: Step 1 (fragmentation), which incubates for 1h 0m 0s
Afternoon: Steps 2-3 (end repair, ligation, and bead clean up)
Freeze overnight at -20°C Day 2
Morning: start Step 4 (bisulfite conversion), which has a 1h 15m 0s incubation at the beginning
Afternoon: continue Step 4 (bead clean up)
Set up Step 5 (PCR) run - hold overnight at 4°C in ThermoCycler or fridge after protocol finishes Day 3
Step 6 (bead clean up and amplification)

Notes:

Do not vortex enzymes or mixtures that contain enzymes.
Handle bisulfite converted DNA with care. Do not vortex or freeze-thaw . The DNA is single stranded, and therefore very fragile.
Label all plates throughout the protocol

Attachments

SMack_Lab_neb_index_primers.xlsx

Steps

Fragment DNA

Prepare fragment master mix (fragment MM) in a 1.5 mL tube for n+1 samples.

Per sample, prepare 4 µL mixture containing:

(Thaw) : 3µL
(On ice) : 1µL

Invert to mix. DO NOT VORTEX.

1.1.

Spin down samples before adding fragment MM. Add 4µL of fragment MM to the template DNA.

Note

Template DNA = 200 ng template DNA + nuclease free H₂O for a total of 26µL

Total volume: 30µL

Cover and spin down samples before incubation.

1.2.

Incubate samples at 37°C for 1h 0m 0s

Note

Do not heat the lid higher than 37°C

SAFE STOPPING POINT : Leave digested DNA in ThermoCycler at 37°C or freeze at -20°C (cover with foil).

Ligation

Prepare end repair master mix (end repair MM) in a 1.5 mL tube for n+1 samples.

Per sample, prepare 5 µL mixture containing:

(On ice) : 1.5µL
(Thaw) : 3.5µL

Invert to mix. DO NOT VORTEX.

2.1.

Add 5µL of end repair MM to each well of fragmented DNA.

Total volume: 35µL

Cover and spin down samples before incubation.

2.2.

Incubate samples for:

0h 30m 0s at 20°C
0h 30m 0s at 65°C
Hold at 4°C

2.3.

Prepare ligation master mix (ligation MM) in a 1.5 mL tube for n+1 samples.

Per sample, prepare 9.25 µL mixture containing:

(Thaw) 1:20 diluted : 1.25µL
Note
Dilute adapters 1:20 in nuclease-free water in a fresh tube
(On ice) : 15µL
(On ice) : 0.5µL

Invert to mix. DO NOT VORTEX.

2.4.

Add 16.75µL of ligation MM to each sample.

Total volume: 51.75µL

Cover and spin down samples before incubation.

2.5.

Incubate for 0h 20m 0s at 20°C

2.6.

Add 1.5µL of to each sample and pipette mix.

Cover and spin down samples before incubation.

2.7.

Incubate for 0h 15m 0s at 37°C

Note

During incubation, take SPRI beads out of fridge to come to room temperature and prepare 50 ml of 80% ethanol.

Bead-Based Cleanup

Add 90µL of Room temperature SPRI beads to each sample and gently pipette mix ~5 times.

3.1.

Incubate at Room temperature for 0h 5m 0s.

3.2.

Place plate on magnetic stand for 0h 5m 0s or until solution is clear.

3.3.

While on the magnetic stand, remove supernatant using a multichannel pipette.

3.4.

While on the magnetic stand:

Add 200µL of 80% ethanol ( do not mix )
Incubate for 0h 0m 30s
Remove ethanol

3.5.

Repeat wash from 3.4

3.6.

Dry the beads for 0h 5m 0s or until beads are no longer shiny.

Note

Be careful to not over dry beads, as this will reduce yield.

3.7.

Remove plate from magnetic stand and add 22µL of EB buffer. Pipette mix and incubate at Room temperature for 0h 5m 0s.

3.8.

Place plate back on magnetic stand and incubate at Room temperature for 0h 5m 0s.

3.9.

Transfer all of supernatant to into a new, sturdy PCR skirted plate.

SAFE STOPPING POINT : Freeze adaptor-ligated DNA at -20°C (cover with foil).

Bisulfite Conversion

Note

This section uses reagents from the Zymo EZ-96 DNA Methylation-Lightning MagPrep kit

Add 130µL of Lightning Conversion Reagent to each 20µL sample and pipette mix.

Total volume: ~150µL

Cover and spin down samples before incubation.

4.1.

Incubate samples for:

0h 8m 0s at 98°C
1h 0m 0s at 54°C
Hold at 4°C (for up to 20h 0m 0s)

4.10.

Incubate at Room temperature for 0h 15m 0s.

While waiting, pre-heat a plate heating element to 55°C. If using a ThermoMixer, put on 96-well attachment.

4.11.

Place the plate on the magnetic stand for 0h 3m 0s or until beads pellet. Remove and discard supernatant.

Note

Important: Take time for handling/re-suspension into account for the total incubation time. Adjust time as necessary to ensure that no sample remains in the L-Desulphonation Buffer for more than 20 minutes.

4.12.

Remove plate from the magnetic stand. Add 200µL of M-Wash Buffer to the beads. Pipette mix 5 times.

4.13.

Place the plate on the magnetic stand for 0h 3m 0s or until beads pellet. Discard supernatant.

4.14.

Repeat M-Wash Buffer wash (steps 4.12-4.13)

Note

Important : Remove as much buffer as possible after final wash to aid in the drying of the beads.

4.15.

Transfer the plate to a heating element at 55°C for 20-30 minutes to dry the beads and remove residual M-Wash Buffer.

Note

Beads will change in appearance from glossy black when still wet to a dull brown when fully dry.

If using the ThermoMixer:

Use the 96-well plate attachment
Rest the deep well plate on top
Check on beads frequently; they may take less than 0h 20m 0s to dry

4.16.

Add 22µL of M-Elution Buffer directly to the dried beads and pipette mix 5-10 times to re-suspend.

4.17.

Heat the elution at 55°C for 0h 4m 0s

4.18.

Transfer the plate to the magnetic stand and incubate at Room temperature for 0h 1m 0s or until beads pellet.

4.19.

Transfer all supernatant into to a new unskirted PCR plate.

Note

It is okay if some beads are removed with the elution.

4.2.

Add 600µL of M-Binding Buffer and 10µL of MagBinding Beads to each well of a 2 mL 96 deep well plate

4.3.

Use multichannel to transfer samples to the 2 mL 96 deep well plate (containing M-Binding Buffer and MagBinding Beads) and pipette mix ~5 times.

4.4.

Incubate at Room temperature for 0h 5m 0s.

4.5.

Transfer plate to a magnetic stand and incubate at Room temperature for 0h 5m 0s or until beads pellet and supernatant is cleared.

4.6.

With the plate on the magnetic stand, remove the supernatant and discard.

Note

Some beads will adhere to the sides of the well. Remove supernatant slowly to allow these beads to be pulled to the magnet as the liquid level is lowered.

4.7.

Remove the plate from the magnetic stand. Add 200µL of M-Wash Buffer to the beads. Pipette mix 5 times.

4.8.

Place the plate on the magnetic stand for 0h 3m 0s or until beads pellet. Remove and discard supernatant.

4.9.

Remove the plate from the magnetic stand. Add 200µL of L-Desulphonation Buffer to the beads. Pipette mix 5 times.

PCR Amplification (Indexing)

Prepare PCR master mix (PCR MM) in a 1.5 mL tube for n+1 samples.

Per sample, prepare 5.625 µL mixture containing:

(Thaw) 5X EpiMark HS Taq Reaction Buffer Catalog #B0490S: 5µL
(Thaw) 10 mM dNTP mix Catalog #N0447S: 0.5µL
(Leave in freezer and add last) EpiMark Hot Start Taq (2 units/ul): 0.125µL

Invert to mix. DO NOT VORTEX.

5.1.

Add 5.625µL of PCR MM to each sample.

Total volume: ~25.625µL

5.2.

Add 1µL of NEBNext Index Primer i7 to each well from the .

5.3.

Add 1µL of NEBNext Index Primer i5 to each well from the and pipette mix.

Note

Important: Ensure all wells are unique combinations .

Cover and spin down samples.

5.4.

Incubate samples for:

95°C for 0h 0m 30s
16 cycles of:

95°C for 0h 0m 15s
61°C for 0h 0m 30s
68°C for 0h 0m 30s

68°C for 0h 5m 0s
Hold at 4°C 0h 5m 0s

Final Cleanup and Quantification

Note

Before you begin, take SPRI beads out of fridge to come to room temperature and prepare 50 ml of 80% ethanol.

Add 50µL of SPRI Beads to each sample. Gently pipette mix.

6.1.

Incubate at Room temperature for 0h 5m 0s.

6.10.

Notes on Pooling:

Find sample with highest DNA conc. and calculate conc. for adding only 1µL to pool
Then, calculate the rest of the samples so that they all have the same concentration going into the pool
Run pool on pippen rep to remove anything under 190 bp
Now, it's ready for sequencing!

6.2.

Place plate on magnetic stand for 0h 5m 0s or until the solution is clear. Remove supernatant.

6.3.

While on the magnetic stand:

Add 200µL 80% ethanol ( do not mix )
Incubate for 0h 0m 30s at Room temperature
Remove ethanol

6.4.

Repeat wash from 6.3

6.5.

After removing the ethanol from the second wash, let the beads dry for 0h 5m 0s or until beads are no longer shiny.

Note

Do not over dry the beads; this will reduce yield.

6.6.

Remove from magnetic stand and add 22µL of nuclease-free H₂O. Pipette mix very well and incubate for 0h 5m 0s at Room temperature.

6.7.

Place tubes back on magnetic stand and incubate for 0h 5m 0s at Room temperature.

6.8.

Transfer all supernatant to new, sturdy skirted PCR plate for long-term storage at -80°C .

Note

If any beads transfer with the supernatant, place plate on magnetic stand when using samples in future protocols.

6.9.

Quantify samples on instrument of choice.

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Reduced Representation Bisulfite Sequencing (RRBS) with NEB Reagents

Abstract

Before start

Attachments

Steps

Fragment DNA

Ligation

Bead-Based Cleanup

Bisulfite Conversion

PCR Amplification (Indexing)

Final Cleanup and Quantification

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