alias: GSM2877235
title: GSM2877235: 3602_WGBS_Olig2; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Genomic DNA was isolated from 1mg to 5mg of tissue sample, using the Qiagen DNeasy Blood and Tissue DNA extraction kit according to manufacturer's instructions. The extracted DNA was suspended in TE buffer and quantified by Qubit (Thermo Fisher Scientific). Libraries were made with In-House Illumina sequencer compatible protocol. The extracted DNA was fragmented by S-series focused ultrasonicator (Covaris) using the “200bp-target peak size protocol”. Fragmented DNA was then size selected (200bp-600bp) with Agencourt aMPure XP bead-based (Beckman Coulter Cat. No. A63880) size selection protocol (Mark. A. Urich et al. 2015). The DNA End repair step was performed with End-It DNA end repair kit (Epicentre, Cat. No. ER81050. After End repair step, A-tailing (NEB, cat. No. M0202) and Ligation steps are performed to ligate methylated adaptors. Bisulfite treatment of genomic DNA was performed using the MethylCode Bisulfite Conversion Kit (Life technologies). Purified genomic DNA was treated with CT conversion reagent in a thermocycler for 10 minutes at 980C, followed by 2.5 hours at 640C. Bisulfite-treated DNA fragments remain Single-stranded as they are no longer complementary. Low-cycle (4-8) PCR amplification was performed with Kapa HiFi Uracil Hotstart polymerase enzyme (KAPA Biosystems, cat. No. KK2801) which can tolerate Uracil residues. The final library fragments contain Thymines and Cytosines in place of the original unmethylated cytosine and methylated cytosines, respectively.
identifiers.namespace: GEO
identifiers.id: GSM2877235