TITLE: Reduced Representation Bisulfite Sequencing (RRBS) with NEB Reagents
AUTHORS: Noah Noah Snyder-Mackler

[DESCRIPTION]
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 60 min
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 75 min 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

[STEPS]
SECTION: Fragment DNA
1. Prepare fragment master mix (fragment MM) in a 1.5 mL tube for n+1 samples.

Per sample, prepare 4 µL mixture containing:
(Thaw)  : 3 µL 
(On ice)  : 1 µL 

Invert to mix. DO NOT VORTEX.
SECTION: Fragment DNA
1.1. Spin down samples before adding fragment MM. Add 4 µL of fragment MM to the template DNA.
 

Total volume: 30 µL 

Cover and spin down samples before incubation.
SECTION: Fragment DNA
1.2. Incubate samples at 37 °C for 60 min 

 


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

SECTION: Ligation
2. 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)   : 1.5 µL 
(Thaw)   : 3.5 µL

Invert to mix. DO NOT VORTEX.
SECTION: Ligation
2.1. Add 5 µL of end repair MM to each well of fragmented DNA.
Total volume: 35 µL 

Cover and spin down samples before incubation.
SECTION: Ligation
2.2. Incubate samples for:
30 min at 20 °C 
30 min at 65 °C 
Hold at 4 °C 
SECTION: Ligation
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   : 1.25 µL 
 

(On ice)   : 15 µL 
(On ice)   : 0.5 µL 

Invert to mix. DO NOT VORTEX.

SECTION: Ligation
2.4. Add 16.75 µL of ligation MM to each sample.
Total volume: 51.75 µL 

Cover and spin down samples before incubation.
SECTION: Ligation
2.5. Incubate for 20 min at 20 °C 
SECTION: Ligation
2.6. Add 1.5 µL of   to each sample and pipette mix.

Cover and spin down samples before incubation.
SECTION: Bead-Based Cleanup
3. 

Add 90 µL of Room temperature SPRI beads to each sample and gently pipette mix ~5 times.
SECTION: Bead-Based Cleanup
3.1. Incubate at Room temperature for 5 min.
SECTION: Bead-Based Cleanup
3.2. Place plate on magnetic stand for 5 min or until solution is clear.

SECTION: Bead-Based Cleanup
3.3. While on the magnetic stand, remove supernatant using a multichannel pipette. 
SECTION: Bead-Based Cleanup
3.4. While on the magnetic stand:
Add 200 µL of 80% ethanol (do not mix)
Incubate for 30 s 
Remove ethanol
SECTION: Bead-Based Cleanup
3.6. Dry the beads for 5 min or until beads are no longer shiny. 
 

SECTION: Bead-Based Cleanup
3.7. Remove plate from magnetic stand and add 22 µL of EB buffer. Pipette mix and incubate at Room temperature for 5 min.
SECTION: Bead-Based Cleanup
3.8. Place plate back on magnetic stand and incubate at Room temperature for 5 min.
SECTION: Bead-Based Cleanup
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). 
SECTION: Bisulfite Conversion
4. 
 


Add 130 µL  of Lightning Conversion Reagent to each 20 µL sample and pipette mix.
Total volume: ~150 µL 

Cover and spin down samples before incubation.
SECTION: Final Cleanup and Quantification
6. 
 

Add 50 µL of SPRI Beads to each sample. Gently pipette mix.
SECTION: PCR Amplification (Indexing)
5. 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: 5 µL 
(Thaw) 10 mM dNTP mix Catalog #N0447S: 0.5 µL
(Leave in freezer and add last) EpiMark Hot Start Taq (2 units/ul): 0.125 µL

Invert to mix. DO NOT VORTEX.
SECTION: Bisulfite Conversion
4.1. Incubate samples for:
8 min at 98 °C 
60 min at 54 °C 
Hold at 4 °C (for up to 1200 min)
SECTION: Bisulfite Conversion
4.2. Add 600 µL of M-Binding Buffer and 10 µL of MagBinding Beads to each well of a 2 mL 96 deep well plate
SECTION: Bisulfite Conversion
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.
SECTION: Bisulfite Conversion
4.4. Incubate at Room temperature for 5 min. 
SECTION: Bisulfite Conversion
4.6. With the plate on the magnetic stand, remove the supernatant and discard.
 

SECTION: Bisulfite Conversion
4.7. Remove the plate from the magnetic stand. Add 200 µL of M-Wash Buffer to the beads. Pipette mix 5 times.
SECTION: Bisulfite Conversion
4.8. Place the plate on the magnetic stand for 3 min or until beads pellet. Remove and discard supernatant.
SECTION: Bisulfite Conversion
4.9. Remove the plate from the magnetic stand. Add 200 µL of L-Desulphonation Buffer to the beads. Pipette mix 5 times.
SECTION: Bisulfite Conversion
4.10. Incubate at Room temperature for 15 min. 

While waiting, pre-heat a plate heating element to 55 °C. If using a ThermoMixer, put on 96-well attachment.
SECTION: Bisulfite Conversion
4.11. Place the plate on the magnetic stand for 3 min or until beads pellet. Remove and discard supernatant.
 

SECTION: Bisulfite Conversion
4.12. Remove plate from the magnetic stand. Add 200 µL of M-Wash Buffer to the beads. Pipette mix 5 times.
SECTION: Bisulfite Conversion
4.13. Place the plate on the magnetic stand for 3 min or until beads pellet. Discard supernatant.
SECTION: Bisulfite Conversion
4.14. Repeat M-Wash Buffer wash (steps 4.12-4.13) 
 

SECTION: Bisulfite Conversion
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.
 

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 20 min to dry
SECTION: Bisulfite Conversion
4.16. Add 22 µL of M-Elution Buffer directly to the dried beads and pipette mix 5-10 times to re-suspend.
SECTION: Bisulfite Conversion
4.17. Heat the elution at 55 °C for 4 min
SECTION: Bisulfite Conversion
4.19. Transfer all supernatant into to a new unskirted PCR plate.
 


SECTION: PCR Amplification (Indexing)
5.1. Add 5.625 µL of PCR MM to each sample.
Total volume: ~25.625 µL

SECTION: PCR Amplification (Indexing)
5.2. Add 1 µL of NEBNext Index Primer i7 to each well from the  .

SECTION: PCR Amplification (Indexing)
5.3. Add 1 µL of NEBNext Index Primer i5 to each well from the   and pipette mix.
 

Cover and spin down samples.
SECTION: PCR Amplification (Indexing)
5.4. Incubate samples for:
1. 95 °C for 30 s
2. 16 cycles of:
95 °C for 15 s
61 °C for 30 s
68 °C for 30 s
3. 68 °C for 5 min
4. Hold at 4 °C 5 min

SECTION: Final Cleanup and Quantification
6.1. Incubate at Room temperature for 5 min.
SECTION: Final Cleanup and Quantification
6.2. Place plate on magnetic stand for 5 min or until the solution is clear. Remove supernatant. 

SECTION: Final Cleanup and Quantification
6.3. While on the magnetic stand:
Add 200 µL 80% ethanol (do not mix)
Incubate for 30 s at Room temperature
Remove ethanol
SECTION: Final Cleanup and Quantification
6.5. After removing the ethanol from the second wash, let the beads dry for 5 min or until beads are no longer shiny.
 

SECTION: Final Cleanup and Quantification
6.6. Remove from magnetic stand and add 22 µL of nuclease-free H2O. Pipette mix very well and incubate for 5 min at Room temperature.

SECTION: Final Cleanup and Quantification
6.7. Place tubes back on magnetic stand and incubate for 5 min at Room temperature.
SECTION: Final Cleanup and Quantification
6.8. Transfer all supernatant to new, sturdy skirted PCR plate for long-term storage at -80 °C .
 


SECTION: Final Cleanup and Quantification
6.9. Quantify samples on instrument of choice.
SECTION: Final Cleanup and Quantification
6.10. Notes on Pooling:
Find sample with highest DNA conc. and calculate conc. for adding only 1 µL 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!
SECTION: Ligation
2.7. Incubate for 15 min at 37 °C
 

SECTION: Bead-Based Cleanup
3.5. Repeat wash from 3.4
SECTION: Bisulfite Conversion
4.5. Transfer plate to a magnetic stand and incubate at Room temperature for 5 min or until beads pellet and supernatant is cleared.
SECTION: Bisulfite Conversion
4.18. Transfer the plate to the magnetic stand and incubate at Room temperature for 1 min or until beads pellet. 
SECTION: Final Cleanup and Quantification
6.4. Repeat wash from 6.3