GSM7311677: Severe_251_Acute; Homo sapiens; Bisulfite-Seq
Isolated PBMCs were subjected to the TRIzol lysis (TRIzol, Thermo Fisher Scientific, Waltham, MA, USA) for paired RNA and genomic DNA isolation. Genomic DNA was isolated from interphase and organic phase using Back Extraction Buffer method (BEB buffer: 4M Guanidine Thiocyanate, 50mM Sodium Citrate NaCl and 1M Tris base prepared in ddH2O) and isopropanol precipitation. The isolated genomic DNA were quantified by PicoGreen dsDNA quantification method. Genomic DNA were converted through sodium bisulfite treatment using DNA Methylation-Direct Kit (Zymo Research) according to the manufacturers' instructions with modifications. Converted genomic DNA were subjected to PCR-free WGBS library construction using extensively optimized PBAT (Post-Bisulfite Adaptor Tagging) for low input and deteriorated genomic materials (OptPBAT). OptPBAT was developed by improving the published PBAT protocols. The fragment sizes of the generated libraries were analysed by Agilent Bioanalyzer. The concentrations of WGBS libraries were estimated using KAPA Library Quantification Kit (KAPA BIOSYSTEM). Libraries were pooled at equimolar ratios into four pools of 20 libraries each and each pool was subjected to an indexed PE sequencing run of 2x151 cycles on one lane of an Illumina NovaSeq 6000 S4 flow cell using custom primers; 5′-GTA AAA CGA CGG CCA GCA GGA AAC AGC TAT GAC-3′ and 5′-GTC ATA GCT GTT TCC TGC TGG CCG TCG TTT TAC-3′.