Illumina HiSeq 3000 paired end sequencing; Bait-capture based single molecule footprinting on ES cells and derivatives
Bait-capture based single molecule footprinting on ES cells and derivatives
ES cells grown under normal conditions were differentiated into neuronal progenitors as described in Bibel et al 2007. In brief, ES cells were swapped into CA medium (ES cell medium with only 10% FCS) for 4 days, followed by 4 additional days in CA medium supplemented with 5micromolar retinoic acid. At day 8, NPs were collected by trypsinization and washing in PBS. Cells are trypsinized, counted and washed in ice cold PBS. For 1 reaction, material equivalent to 250.000 cells were pelleted and resuspended in 1mL ice cold hypertonic buffer (10mM Tris (pH = 7.4), 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA, 0.5% NP40) to release the nuclei, span down and washed in the same buffer without NP40, eliminating detergents. Nuclei were then span down.in 1x M.CviPI reaction buffer (50mM Tris (pH 8.5), 50mM NaCl, 10mM DTT). The nuclei were then incubated with 200U of M.CviPI (NEB-M0227L) at 37C for 7.5 min (in presence of 0.6mM SAM, and 300mM Sucrose). The reaction was supplemented with 100U of M.CviPI and 128pmol of SAM before a second incubation round of 37C for 7.5 min. For ES samples, reactions were stopped at this point by adding a SDS containing buffer (20mM Tris, 600mM NaCl, 1%SDS 10mM EDTA).  For TKO samples, where dual enzyme footprinting was applied, 10mM MgCl2, 128pmol of SAM and 60U of M.SssI (NEB- M0226L) were added for a third incubation round of 37C for 7.5 min followed by stoppage of the reaction by adding the same SDS containing buffer. Samples were then incubated at 55C over night, followed by phenol / chloroform extraction of DNA. 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.