title: GSM4928848: β-actin knockout Mouse Embryonic Fibroblast_IP1; Mus musculus; ChIP-Seq
library_construction_protocol: Mouse Embryonic fibroblasts were crosslinked using 1% formaldehyde (Sigma cat. No. F8775) for 10 minutes. Quenching was carried out using 0.125 M Glycine for 5 minutes and cells were then lysed using lysis buffer 1 -LB1-(50 mM Hepes KOH pH 7.5, 10 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP–40, 0.25% Triton X-100). Nuclei were pelleted, collected and washed using lysis buffer 2 -LB2- (10 mM Tris HCl pH 8, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA). This was followed by lysis using lysis buffer 3 LB3 (10 mM Tris HCl pH 8; 100 mM NaCl, 1mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate, 0.5% N-laurylsarcosine). Chromatin was sheared using Qsonica Sonicator (4 cycles of 3 minutes at 70 % Amplitude), and then checked on 0.8% agarose gel. 100 μg of fragmented chromatin was mixed with antibody. The protein-antibody immunocomplexes were recovered by the Pierce Protein A/G Magnetic Beads (Thermo-Scientific). Beads and attached immunocomplexes were washed twice using Low salt wash buffer (LS) (0.1% SDS; 2mM EDTA, 1% Triton X-100, 20 mM Tris HCl pH8, 150 mM NaCl), and High Salt (HS) wash buffer (0.1% SDS, 2mM EDTA, 1% Triton X-100, 20 mM Tris HCl pH8, 500 mM NaCl) respectively. The beads were re- suspended in elution buffer (50 mM Tris HCl pH8, 10 mM EDTA, 1% SDS). De-crosslinking was achieved by adding 8µl 5 M NaCl and incubating at 65°C overnight. RNase A (1 µl 10 mg/ml) was added for a 30 min incubation at 37°C. Then, 4 µl 0.5 M EDTA, 8 µl 1 M Tris-HCl, and 1 µl 20 mg/ml proteinase K (0.2 mg/ml) were added for a 2 hours incubation at 42°C to digest the chromatin. DNA was purified by QIAquick PCR purification kit (Qiagen, Germantown, MD, USA) for qPCR analysis and sequencing. ChIP-seq library preparation was done using the TruSeq Nano DNA Library Prep Kit (Illumina, San Diego, CA, USA) and then sequenced with the NextSeq550 sequencing platform (performed at the NYUAD Sequencing Center).
attributes.tag: GEO Accession
attributes.value: GSM4928848