title: GSM4680421: HeLa_shINTS11_ctrl_r2_RNAPII-Ser2_ChIP-seq; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-seq was performed as previously described (Gardini, A. et al. 2014, Lai, F. et a.l 2015, Yue, J.  et. al 2017) with slight modifications. 2 x 107 cells were crosslinked in 1% formaldehyde for 10 minutes at room temperature and quenched with 125 mM glycine. Samples were resuspended in ChIP lysis buffer (20 mM Tris-HCl, 50 mM NaCl, 0.1% SDS, 0.5% Triton-X-100, 1 mM EDTA) and sonicated in a Covaris M220 focused sonicator until chromatin fragments of 150-300 bp were produced. Protein A/G magnetic beads were bound with antibody and incubated with 1 mg of sonicated chromatin overnight. The following day, beads were washed twice with Mixed Micelle Buffer (150 mM NaCl, 1% Triton X-100, 0.2% SDS, 20 mM Tris-HCl, 5 mM EDTA, 65% sucrose), Buffer 500 (500 mM NaCl, 1% Triton X-100, 0.1% Na deoxycholate, 25 mM HEPES, 10 mM Tris-HCl, 1 mM EDTA), LiCl/detergent wash (250 mM LiCl, 0.5% Na deoxycholate, 0.5% NP-40, 10 mM Tris-HCl, 1 mM EDTA), and eluted in Decrosslinking Buffer (20 mM Tris-HCl, 300 mM NaCl, 0.5% SDS, 1 mM EDTA) at 65 °C. Eluates were treated with RNAse A for 1 hour at 37 °C and then Proteinase K for 3 hours at 56 °C. The DNA was finally purified by phenol/chloroform and eluted in 1x TE. ChIP-seq libraries were generated using the NEBNext Ultra II DNA library prep kit (New England Biolabs, #E7645S) with at least 10 ng of input DNA.
attributes.tag: GEO Accession
attributes.value: GSM4680421