GSM4675256: ES cells_DNA_rep2; Mus musculus; Bisulfite-Seq
DNA was extracted after each treatment timepoint using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Methyl-seq was performed by Genomics, Epigenomics and Sequencing Core at the University of Cincinnati. SureSelect Methyl-Seq Target Enrichment System for mouse (Agilent, Santa Clara, CA) was used to prepare the sequencing library. A total of 1 μg high quality genomic DNA quantified by Qubit assay (Thermo Fisher, Waltham, MA) was sheared by Covaris S2 focused-ultrasonicator (Covaris, Woburn, MA) to about 200 bp under the recommended settings, and validated by 2100 Bioanalyzer (Agilent, Santa Clara, CA). Next, the DNA fragments were end prepared and ligated to the methylated adaptor. The size of the ligated libraries was then validated by Bioanalyzer, followed by hybridization with biotin-labeled RNA-baits to capture the regions where methylation is known to impact gene regulation. After hybridization, libraries were captured with streptavidin beads and bisulfite modified with EZ DNA Methylation-Gold kit (Zymo, Irvine, CA), and enriched with 8 cycles of PCR. These individually amplified libraries were labeled with unique indices by 6 cycles of PCR. After purification and size selection of the indexed libraries using AMPure XP beads (Beckman Coulter, Indianapolis, IN), the quality and quantity of the libraries were assessed by Bioanalyzer High Sensitivity DNA assay. To quantify the library concentration for the clustering generation, the libraries were qPCR analyzed by NEBNext Library Quant Kit (New England BioLabs, Ipswich, MA) using QuantStudio 5 Real-Time PCR System (Thermo Fisher, Waltham, MA). The sequencing was performed using Illumina Nextseq 550 sequencer under the sequencing setting of PE 2x150 bp to generate ~60M pass filter reads per sample.