GSM5050205: SA03907_S35; Homo sapiens; Bisulfite-Seq
Genomic DNA was extracted from whole blood samples using QIAGEN's QIAamp DNA Blood Midi/Maxi Kit, performed at Duke. The purified genomic DNA was shipped on ice to University of California, Los Angeles (UCLA) for RRBS library preparation. Genomic DNA was first quantified by Qubit dsDNA BR Assay Kit (ThermoFisher cat. Q32850). The RRBS libraries were prepared as described(52) with the following conditions. For each sample, 100 ng of purified genomic DNA was digested with 20 U of MspI (NEB, cat # R0106L) at 37°C o/n in the presence of RNase Cocktail Mix (Ambion, cat # AM2286). End-repair and adenylation tailing were performed by the addition of Klenow Fragment 3ʹ->5ʹ exo- (NEB, cat # M0212L) in the presence of dATP, dGTP, and d5meCTP (Fermentas). Adapter Ligation was performed by the addition of 0.3 µl of Illumina TruSeq methylated Adapters (Illumina, TruSeq Nano cat# FC-121-4001) and 1 µl of Quick T4 DNA Ligase (NEB cat# M2200L). Samples were pooled and purified using an equal volume of SPRI beads (Beckman Coulter, cat # B23318). Size-selection was performed using SPRI beads to enrich for fragments from 200 to 400 bp. Bisulfite treatment was performed using EpiTect Bisulfite kit (QIAGEN, cat # 59104) according to manufacturer's protocol, except that two consecutive rounds of conversion were performed, for a total of 10 hr of incubation. Purified converted DNA was PCR amplified using MyTaq HS Mix (Bioline, cat# BIO-25045) and TruSeq PCR Primer Cocktail (Illumina, TruSeq Nano cat# FC-121-4001) according to the following protocol: initial denaturation at 98°C for 30 s; 12 cycles of 98°C for 15 s, 60°C for 30 s, 72°C for 30 s; final extension at 72°C for 5 min. Amplified libraries were purified twice with an equal amount of SPRI beads to remove primer and adapter dimers. The sequencing data were first demultiplexed based on the barcodes of each sample pooled in the lanes. The data were subjected to a quality control step using FastQC (version 0.11.7), where the low quality reads were discarded from further analysis based on the per base sequence quality and per sequence quality score using the default criteria setting in FastQC(53). Sequencing data were trimmed to remove the adaptors and the terminal CG using the trimmomatic software (v0.38) (54). The reads were aligned to the human genome GRCh38/hg38 (release 93, downloaded from Ensembl) and DNA methylation levels were determined using the BS-Seeker2 pipeline(55) with the aligner bowtie (v1.2.2) and the default settings for the fragment length (20 for lower bound, 500 for upper bound). Samples with less than 5 M total raw reads (3 samples) were excluded from further analyses. On average each sample generated about 36 M total raw reads with 73.7% mappability. Within the study, sequencing data were generated from two sequencing runs, termed batch.