Reduced Representation Bisulfite Sequencing (RRBS) Protocol

MspI Digestion of Genomic DNA

1. Add 100 ng (in 25 μL) of genomic DNA to a 0.2-mL tube, and keep the eight-tube strip on ice.
2. Prepare a master mix of 10 Buffer Tango (3 μL) and MspI (10 U/μL) (2 μL), and keep the master mix on ice. Add 5 μL of the master mix to the 0.2-mL tube containing genomic DNA. Mix by pipetting and cap the eight-tube strip with an eight-cap strip.
3. Place the eight-tube strip (0.2 mL) on a thermal cycler, and run the following thermal program for MspI digestion: 37°C for 120 min; hold at 4°C (heated lid 50°C).

End Repair and dA-Tailing

1. Spin down the eight-tube strip (0.2 mL) containing MspIdigested DNA. Remove the eight-cap strip carefully without splashing the DNA solution in the tubes. Keep the eight-tube strip on ice. Add 1 μL of dNTP mix for RRBS and 1 μL of Klenow Fragment (exo-) to each tube and mix by pipetting (32 μL in total). Cap the tubes with a new eight-cap strip.
2. Place the eight-tube strip (0.2 mL) on a thermal cycler, and run the following thermal program: 30°C for 20 min and 37°C for 20 min; hold at 4°C (heated lid 50°C).
3. Add 64 μL (2 volumes) of AMPure XP beads to the DNA solution. Perform AMPure XP beads purification, and elute DNA with 18 μL of 10-mM Tris–Cl (pH 8.5). Transfer 17.7 μL of the eluted DNA solution to a new eighttube strip (0.2 mL).

Adaptor Ligation

1. Keep the eluted DNA in the eight-tube strip (0.2 mL) on ice. Add 2.3 μL of NEBNext Ultra II End Prep Reaction Buffer to the eluted DNA (20.0 μL in total).
2. Prepare a master mix for NEBNext Ultra II Ligation Master Mix (10.0 μL) and NEBNext Ligation Enhancer (0.33 μL), both of which are included in reagent 5 in Subheading 2.1. Mix the master mix by pipetting up and down ten times. Add 10.33 μL of the master mix to eluted DNA and mix by pipetting up and down three times. Add 0.83 μL of NEBNext Methylated Adaptor (2 μM) to each tube, and mix by pipetting up and down ten times (31.16 μL in total). Cap the eight-tube strip with a new eight cap strip.
3. Place the eight-tube strip on a thermal cycler, and run the following thermal program: 20 °C for 60 min; hold at 4 °C (heated lid 50°C). After removing the eight-cap strip, keep the eight-tube strip on ice. Add 1 μL of UracilSpecific Excision Reagent Enzyme, mix by pipetting, and cap the eight tube strip with a new eight-cap strip. Place the tubes on a thermal cycler, and run the following thermal program (Program D): 37°C for 15 min; hold at 4 C (heated lid 50 °C). Store the tubes containing adaptor-ligated DNA at 20 °C until the next day.
4. Add 48 μL (1.5 volumes) of AMPure XP beads to the DNA solution. Perform AMPure XP beads purificatio, and elute DNA with 20 μL of 10-mM Tris–Cl (pH 8.5).

Bisulfite Conversion of Adaptor-Ligated DNA

1. Perform bisulfite conversion of adaptor-ligated DNA using EZ Methylation Gold Kit. The thermal program required for bisulfite conversion is 98°C for 10 min and 64°C for 150 min, and hold at 4 °C (heated lid 105°C). Add 130 μL of CT conversion reagent to the adaptor-ligated purified DNA (20 μL), and divide the solution into two 0.2-mL tubes (75 μL each). Place the eighttube strip(s) on a thermal cycler and run. After its completion, combine the two aliquots of 75 μL in one tube. Conduct the subsequent procedures according to the manufacturer's protocol of the EZ Methylation Gold Kit. Elute bisulfite-converted DNA with 11 μL of M-Elusion Buffer.

PCR Amplification

1. Transfer 10.0 μL of bisulfite-treated DNA to a 0.2-mL tube. Add 1.25 μL of i7 primer (10 μM) and 1.25 μL of i5 primer (10 μM) to the bisulfite-treated DNA, and mix briefly. Add 12.5 μL of 2 KAPA HiFi Hot Start Uracil+ Ready Mix and mix by pipetting (25.0 μL in total).
2. Conduct PCR amplification using the following thermal cycling conditions (Program F): 98°C for 45 s for the initial denaturation, nine cycles of 98°C for 15 s, 60°C for 30 s, and 72°C for 30 s for amplification and 72°C for 1 min for final extension; hold at 4°C (heated lid 105°C). The amplified DNA samples can be kept overnight at 4°C in a thermal cycler or kept longer at 20°C.

Library Quality Control

1. When the High Sensitivity DNA Kit is used, 1 μL each of the purified RRBS libraries is subjected to the microcapillary-based electrophoretic analysis.
2. Measure the molarity of each library (for the range 200 bp–1000 bp). Check the fragment size distribution of each library.

Sequencing

1. Test sequencing on the MiSeq platform and readjustment of library DNA concentration: Prepare more than 2 μL of 2-nM solution for each library. Pool 2 μL each of the 2-nM libraries (a total of 16 μL in case of eight libraries). For a test sequencing using a MiSeq Reagent Nano Kit v2 (300-cycles), mix 92 μL of 10-mM Tris–Cl (pH 8.5), 2 μL of the 2-nM library pool, and 1 μL of 1-nM PhiX Control v3 Library (95 μL in total) in a 1.5-mL tube. Add 5 μL of 2-N NaOH to the 95-μL mixture, mix by vortexing, and leave the tube at room temperature for 5 min to denature the library DNA mixture. Add 500 μL of ice-cold Buffer A, mix, and keep the solution on ice for 5 min. The final concentration of the denatured library solution (600 μL in total), including 20% amount of spike-in PhiX Control v3 Library for color balancing of low nucleotide diversity, is 8.3 pM. Run the test sequencing on a MiSeq platform using the Generate FASTQ workflow.
2. After the completion of the MiSeq run, check the index ratios of the RRBS libraries, and recalculate the concentration of the libraries in accordance with the index ratios.
3. Conduct full-scale sequencing on the HiSeq X platform. Prepare a pool of RRBS libraries sufficient for sequencing.