title: GSM8006460: WGBS-Ogt_iKO mESC, +4OHT 6-days, rep#2; Mus musculus; Bisulfite-Seq
library_name: GSM8006460
library_construction_protocol: In order to ensure a high purity of our samples, we isolate specifically the YFP+ population (in the case of 4-OHT-treated Ogt iKO mESC) at the same time that eliminating the presence of mitomycin-C-treated MEFs (used as feeders during our mESC cultures) by FACS (Fluorescence-Activated Cell Sorting) using a FACSAria cell sorter (BD Biosciences). The dissociated mESC were first labeled with the eBioscienceTM Fixable Viability Dye eFluorTM 780 (ThermoFisher, 65- 0865-14) to select viable cells and then stained for surface markers with indicated fluorochrome-conjugated antibodies diluted in FACS buffer (PBS 1X, 0.5% BSA, 0.01% NaN3): CD90.2 (Biolegend, 140319) to positively distinguish the mESC and CD326 (Biolegend, 118227) to negatively exclude MEFs. Genomic DNA was isolated using a genomic DNA mini kit or FlexiGene DNA Kit (Qiagen, 51206). Unmethylated lambda DNA (Promega, D1521) was spiked into the genomic DNA samples at a ratio of 1:200 to monitor the bisulfite conversion efficiency. The samples were then sheared using Covaris S2, purified with Ampure XP beads (Beckman Coulter) and processed with end repair and dA-Tailing Modules, and ligated to methylated Illumina Adaptors using a quick ligation module. DNA with ligated adaptors was then treated with sodium bisulfite for 4hrs and amplified with index primers using KAPA HiFi HotStart Uracil+ Ready Mix (KAPA Biosystems).