GSM2498117: C554_ACTTGA_L003_R1; Homo sapiens; Bisulfite-Seq
Genomic DNA was extracted from human blood using the DNeasy Blood and Tissue Kit from Qiagen (Valencia, CA), mixed well, quantified via NanoDrop (Wilmington, DE), and then aliquoted into samples for rapid multiplexed reduced representation bisulfite sequencing (rmRRBS) Briefly, we prepared the RRBS libraries as follows. First, we digested200ng of gDNA overnight at 37oC using 10U of MspI restriction endonuclease (C↓CGG) from New England Biolabs (NEB; Ipswitch, MA) generating fragments that each contain at least two CpG loci. We next filled in the 3’ overhangs and added an adenosine residue to each terminus of the DNA fragments via end repairing and A-tailing reagents from NEB. We then ligated indexed, methylated Illumina TruSeq adapters to the A-tailed fragments using a T4 ligase from NEB. All adapters contain universal PCR primer and NGS sequencing primer sequences, but the different adapters have unique six base index sequences, sometimes referred to as barcodes, that allow multiple different RRBS libraries to be sequenced in a single lane and then distinguished bioinformatically after sequencing. We then selected adapter-ligated DNA fragments larger than 100bp using positive selection with 1.2X volume of AMPure XP magnetic beads from Beckman Coulter, which also cleaned up the mRRBS libraries. We next pooled either 6 or 12 libraries, and then treated the pools with sodium bisulfite using the EZ DNA Methylation Gold kit from Zymo according to the manufacturer's instructions. Bisulfite converted library pools, which now contained uracil in place of unmethylated cytosines, were then PCR amplified using the minimum number of cycles (determined using a small scale test reaction with each pool) with a Pfu Turbo Cx Hotstart DNA polymerase from Agilent Technologies. We then cleaned up the final amplified library pools using a 1.8X volume of AMPure XP magnetic beads. mRRBS libraries were sequenced on an Illumina HiSeq2500 using 75 base-pair single-end reads. All libraries were sequenced at normal cluster density except those prepared from participants C and D, as well as a replicate prepared from participant E, which were run at high cluster density.