GSM3770659: OISD5_siDNMT1; Homo sapiens; Bisulfite-Seq
The genomic DNA was isolated by using DNeasy Blood & Tissue Kit. Two micrograms of genomic DNA were spiked with unmethylated bacteriophage λ DNA (5 ng of λ DNA per microgram of genomic DNA; Promega) and with methylated T7 phage DNA (5 ng of T7 DNA per microgram of genomic DNA). The DNA was sheared to 50–500 bp in size using Covaris LE220 ultrasonicator, and fragments of 150–300 bp were size-selected using AMPure XP beads (Agencourt Bioscience). The libraries were constructed using the KAPA Library Preparation Kit with no PCR Library Amplification/Illumina series (Roche-Kapa Biosystems). After adaptor ligation, NEXTFLEX® Bisulfite-Seq Barcodes (Perkin Elmer), the DNA was treated with sodium bisulfite using the EpiTect Bisulfite kit (Qiagen) following the manufacturer's instructions. Enrichment for adaptor-ligated DNA was carried out through seven PCR cycles using KAPA HiFi HotStart Uracil+ReadyMix PCR 2x Kit (Roche-Kapa Biosystems). Library quality was monitored using the Agilent 2100 Bioanalyzer DNA 7500 assay, and the library concentration was estimated using quantitative PCR using the KAPA Library Quantification Kit for Illumina® Platforms, v1.14 (Roche-Kapa Biosystems).  Paired-end DNA sequencing (2×101 bp) of the oxBS and BS libraries was performed using the HiSeq2000 (Illumina) with TruSeq SBS Kit v3-HS following the manufacturer's protocol with HiSeq Control Software (HCS) 1.5.15.1 or using TruSeq SBS and flow cell kit v4. Images analysis, base calling and quality scoring of the run were processed using the manufacturer's software Real Time Analysis (RTA 1.13.48 for v3 and RTA 1.18.66.3 for v4) and followed by generation of FASTQ sequence files.
GEO Accession
GSM3770659