title: GSM4654798: F-6_day_70; Homo sapiens; Bisulfite-Seq
library_construction_protocol: AllPrep DNA/RNA isolation kit according to the manufacturer's instructions (Qiagen) Genomic DNA isolated from patient PBMC samples was bisulfite converted using the EZ DNA Methylation- Gold Kit (Zymo Research) according to the manufacturer's instructions.  Libraries were prepared using the NEBNext Ultra II DNA kit (NEB) according to manufacturer instructions with few modifications. Briefly, sonicated DNA was subjected to EndPrep (End Repair and A-tailing), followed by Adapter Ligation using 2.5μL of Illumina TruSeq pre-methylated Adapters (Illumina). Samples were purified using 0.85x NEB Purification Beads and eluted in 15μL of 10mM Tris-HCl, pH 8. Samples were mixed in 16-sample pools and column purified using a DCC-5 (Zymo Research). Elution was performed with 10μL of 60°C 10mM Tris-HCl, pH 8. Each sample pool was subject to hybrid capture with custom biotinylated RNA probes designed to tile the MSCV vector (MyBaits Arbor Bioscience - Human_6K and Human patch2) according to the manufacturer's protocol. Captured DNA was eluted by heating in 20μL of 10mM Tris-HCl  pH 8+0.05% Tween-20. The eluted DNA was then subject to bisulfite conversion using the DNA Methylation  Lightning kit (Zymo Research). Converted DNA was then amplified using xGen Library Amplification Primer  Mix (IDT) and Kapa Uracil+ Ready Mix.