GSM5746733: Maz KO ESC, CTCF ChIP, rep2; Mus musculus; ChIP-Seq
In the case of samples containing DSG, they were crosslinked with 2mM DSG in 1X PBS for 30min at RT, and followed by 1% formaldehyde in DMEM for 10min. For the rest of the samples, crosslinking was performed with 1% formaldehyde in DMEM for 10min. Cells were lysed as described in Oksuz et al., 2018 using buffers in the following order: LB1 (50 mM HEPES, pH 7.5 at 4 C, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton X) for 10 min at 4 C, LB2 (10 mM Tris, pH 8 at 4 C, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 10 min at RT, and LB3 (10 mM Tris, pH 7.5 at 4 C, 1 mM EDTA, 0.5 mM EGTA, and 0.5% N-Lauroylsarcosine sodium salt). Chromatin was sonicated to an average size of ~250 bp using a Diagenode Bioruptor. ChIP was performed by using antibodies listed. Chromatin from Drosophila (1:100 ratio to ESC or MN derived chromatin), and Drosophila-specific H2Av antibody were used as spike-in control in each sample. Libraries were prepared according to manufacturer's instructions (Illumina) as described in Narendra et al., 2015. Immunoprecipitated DNA was first end-repaired using End-It Repair Kit (Epicenter), tailed with an A using Klenow exo minus (NEB M0212), ligated to custom adapters, and fragments of 300±100 bp were size-selected. These fragments were amplified through ligation-mediated PCR amplification (LM-PCR) by using Q5 polymerase (NEB M0530).