GSM5708745: CIC-WT (IDH1-R132H) WGBS rep 1; Homo sapiens; Bisulfite-Seq
Whole genome bisulfite sequencing was performed on two replicates per each cell line. To track the efficiency of bisulphite conversion, 10 ng lambda DNA (Promega) was spiked into 1 µg genomic DNA, quantified using Qubit fluorometry, and arrayed in a 96-well microtitre plate. DNA was sheared to a target size of 300 bp using Covaris sonication (2 x 50 s at a “Duty cycle” of 30%, 450 Peak Incident Power [W] and 200 Cycles per Burst) and the fragments were subject to end-repair and phosphorylation in a single reaction, using an enzyme premix (NEB) containing T4 DNA polymerase, Klenow DNA Polymerase and T4 polynucleotide kinase, and incubated at 20 °C for 30 min. Repaired DNA was purified in 96- well format using PCR Clean DX beads (Aline Biosciences), and 3'-A-tailed (adenylation) using Klenow fragment (3' to 5' exo minus) at 37 °C for 30 min prior to enzyme heat inactivation. Cytosine methylated paired-end adapters: (5'- mCAmCTmCTTTmCmCmCTAmCAmCGAmCGmCTmCTTmCmCGATmCT-3'