knockdown for DNA methyltransferases DNMT1, DNMT3A, DNMT3B
Sequenced DNA Library
ES cell samples were washed 2x (in KO medium), counted to normalize by cell number, cross-linked (ten minutes rotation in 1% formaldehyde at RT), quenched with glycine at 125mM, incubated 5' on ice, washed 3x (cold PBS) and pelleted at 10e7 cells per eppendorf. Pellets were lysed according to Covaris protocol: pellets were resuspended in ice cold buffer 1 (50mM HEPES-KOH pH7.4, 140mM NaCl, 1mM EDTA, 0.5mM EGTA, 10% glycerol, 0.5% NP40, 0.25% TX100, antiproteases), then in ice cold buffer 2 (10mM Tris pH8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, antiproteases) then washed 3 times and resuspended in 1ml sonication buffer on ice (10mM Tris pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% NaDOC, 0.25% NLS and protease inhibitors), transferred to TC12 glass tubes (Covaris: 520056) and sonicated (Covaris settings: 20% duty cycle, intensity 5, 200cyles/ burst, 15, 30 or 60 minutes). Sonication was then assessed by reverse cross-linking overnight in the presence of proteinase K and RNase, followed by DNA extraction and quantification and quality control on a Bioanalyzer (Agilent 2100). Samples were also checked for the absence of single-stranded DNA by Exonuclease I treatment and the 60' sonicated samples were used for further chromatin immunoprecipitation. Paired-end libraries were prepared and sequenced in 100bp reads runs according to Illumina instructions.