GSM6560760: Cyclin B1 -ve [C04]; Homo sapiens; Bisulfite-Seq
Cells were glyoxal-fixed and stained for CCNB1 as previously described (Channathodiyil and Houseley, 2021) and single cells sorted by flow cytometry to collect CCNB1 positive and CCNB1 negative cells in a 96-well plate. Bisulfite conversion was carried out using EZ Methylation Direct bisulfite reagent (Zymo) and desulfonation and purification of converted DNA with magnetic beads (Zymo) on the Bravo automated workstation (Agilent) with DNA eluted into the master mix for the first-strand synthesis. Random priming and extension were performed followed by purification of the resulting DNA fragments. Second strand synthesis was then performed and the resulting fragments amplified with 14 PCR cycles. Amplified libraries were pooled (2 pools of 48 libraries each) and purified using 0.7× Ampure XP beads. Single cell bisulfite libraries were then prepared as (CLark et al 2017)