title: GSM4513840: 4Cseq_of_BHLHE40_Mit_3Hours_rep1; Mus musculus; OTHER
library_construction_protocol: 2 million cells were crosslinked in 1% formaldehyde at RT for 10 min and quenched with 125 mM glycine for 5 min. The cell pellets were washed twice in PBS and resuspended in 300μl lysis buffer (10mM Tris-HCl (pH 8.0), 10mM NaCl, 0.2% Igepal CA630 (Sigma I8896)), then incubated on ice for 20 min. Following centrifugation at 2500xg for 5 min at 4°C, the pellet was resuspended in 50uL of 0.5% SDS and incubated for 10 min at 65°C. SDS was quenched with 145uL water and 25uL of 10% TritonX-100 for 15 mins at 37°C. 25ul of CutSmart buffer (NEB B7204S) was added with 10ul DpnII enzyme (NEB R0543M) and the samples were incubated overnight at 37°C with 700rpm rotation. After confirming the digestion efficiency, the enzyme was inactivated by incubating at 65ºC for 20 mins. The samples were then diluted with 669μl Milli-Q water, 120μl T4 ligation buffer (NEB B0202), 60μl 10mM ATP (NEB P0756S), 120μl 10% Triton X-100, 6μl 20mg/ml BSA and 5μl 400U/μl T4 DNA Ligase (NEB M0202) for 3h on a rotor at RT. The samples were then treated with proteinase K and reverse crosslinked overnight. Following RNAse treatment, phenol/chloroform extraction and DNA precipitation, the pellets were dissolved in 100μl of 10mM Tris pH 8. The second digestion was performed by adding 20μl 10x buffer B (Fermentas), 10μl Csp6I (Fermentas, ER0211), 80μl milli-Q water, and incubating overnight at 37°C with 700rpm rotation. After confirming the second digestion efficiency, the enzyme was again inactivated. Another ligation was performed by adding 300μl T4 ligation buffer, 150μl 10mM ATP, 5μl T4 DNA Ligase, and Milli-Q water to 3mL and incubating overnight at 16°C. The DNA was purified by phenol/chloroform extraction and ethanol precipitation, resulting in the 4C-template. 4C-seq
attributes.tag: GEO Accession
attributes.value: GSM4513840