GSM1505751: SMAD4 MNChIP-seq in cell type HUES64 Smad4 021113 h64; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Pluripotent stem cell
Attributes by original data submitter
Sequenced DNA Library
Cells were grown to a final count of 10 million, resuspended in PBS, and crosslinked in 10% formaldehyde solution for 10 minutes at room temperature. Following quenching with 0.125M glycine and two PBS washes, we isolated nuclei using cell lysis buffer (20 mM Tris-HCl ph8, 85mM KCl, 0.5% NP40). Nuclei were then digested using MNase (Worthington, LS004797) as done in (Henikoff et al., PNAS 2011). Digestion was stopped with 0.05M EGTA and chromatin was aliquoted into 1-2 million cells per ChIP. Antibodies were added and immunoprecipitation was carried out overnight at 4°C as done in1. The next day, protein G beads (Life Technology, 10009D) were added for 2 hours at 4°C to isolate the protein bound DNA and washed twice using Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 150mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 500mM NaCl), LiCl Wash Buffer (0.25M LiCl, 0.5% NP40, 0.5% sodium deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1,), and TE Buffer pH 8 (10mM Tris-HCl, pH 8, 1mM EDTA pH 8). DNA was eluted twice using 100µL of ChIP Elution Buffer (1% SDS, 0.1M NaHCO3) at 65°C for 15 minutes. Crosslinking was reversed by addition of 32µl reverse crosslinking salt mixture (250 mM Tris-HCl pH 6.5, 62.5 mM EDTA pH 8, 1.25 M NaCl, 5mg/ml Proteinase K) for 5-18 hours at 65°C. DNA was isolated using phenol/chloroform extraction and treated with DNase-free RNase for 30 minutes at 37°C. The whole cell extract (WCE) control was generated using MNase treated material that was then reverse crosslinked and phenol chloroform extracted, skipping the immunoprecipitation and washing steps. MNChIP-seq library construction was performed as in (Henikoff et al., PNAS 2011) with gel size selection from 140bp to 720bp as described in (Gu et al., 2010). RRBS was performed according to a previously published protocol (Smith et al., 2009) with some optimizations for small cell numbers (Gu et al., 2010).