Bait-capture based single molecule footprinting in 159 DNMT TKO mESCs upon p300 inhibition
Bait-capture based single molecule footprinting in 159 DNMT TKO mESCs upon p300 inhibition
Mouse ES cells were cultured on 0.2% gelatin-coated plates in ES medium (DMEM, supplemented with 15% FBS, LIF, 2-Mercaptoethanol, 2 mM L-Glutamine and 1x non-essential amino acids) at 37C and 5% CO2. Medium was changed daily and cells were split every second day. p300 inhibition was performed by adding 3 Î¼M A-485 (Selleck Chemicals, S8740) to the ES medium for 24 hours. Genome-wide data was obtained using Agilent SureSelectXT Mouse Methyl-Seq Kit.  The library preparations, for the genome-wide data, were performed according to the SureSelect XT Mouse Methyl-Seq Kit Enrichment System for Illumina Multiplexed Sequencing Library protocol (Agilent Technologies, Santa Clara CA, Version E0, April 2018). A total of 3 microg of footprinted DNA was used as input for bait capture, according to the company's specifications. (# 5190-4836). DNA was first sonicated using a Covaris S220 sonicator to obtain products of 200-300 bp. DNA was then end-repaired, A-tailed and ligated with methylated adapters to create a pre-capture DNA library. Adapter-ligated libraries were purified using (0.65X) AMPure XP beads then quality and quantity of libraries were determined by bioanalyzer using DNA high sensitivity chip (Agilent). Next, 350 nanog of each library was hybridized with the SureSelect Mouse methyl-seq capture library at 65 C for 16 hours. Hybridized products were purified by capture with Dynabeads MyOne Streptavidin T1 magnetic beads and then subjected to bisulfite conversion using EZ DNA Methylation- Gold Kit kit according to manufacturer's protocol. As described in manufacturer's protocol (Agilent SureSelectXT Mouse Methyl-Seq Kit), bisulfite converted libraries were PCR-amplifed for 8 cycles with supplied universal primers and purified using AMPure XP beads. Captured libraries were indexed by PCR for another 6 cycles, using supplied indexes to generate a multiplexed library. High quality libraries were identified with an Agilent bioanalyzer using DNA high sensitivity chip and then pooled for sequencing. Supplied primers and recommended amplification parameters of the manufacturer were used throughout library preparation.