GSM1847369: ABL_Pol2_siCon_FP_inhibition_40min_ChIPSeq; Homo sapiens; ChIP-Seq
cells were grown to 70%-80% confluence, and crosslinked with 1% formaldehyde for 10 min at room temperature. After washing twice with cold PBS, cells were collected and resuspended in lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris (pH 8.0), 1x protease and phosphatase inhibitors). After sonication, the soluble chromatin was diluted in 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris (pH 8.0), 1x protease and phosphatase inhibitors). Then samples were precleared and immunoprecipitated with the appropriate antibodies overnight at 4°C. The next day, protein A or G beads blocked with BSA were added for 2 h at 4°C, and then washed sequentially for 10 min with TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8.0), 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8.0), 500 mM NaCl), buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris (pH 8.0), and TE buffer (10 mM Tris (pH 8.0), 1mM EDTA (pH 8.0)). DNA was eluted in Elution buffer (1% SDS, 0.1M NaHCO3) at room temperatue for 30 min. The crosslinking was reversed by incubation for 16 h at 65°C. During the purification, ChIP DNA was treated with DNase-free RNase for 30 min at 37°C. Libraries were prepared according to NEBNext ChIP-Seq Library Prep Master Mix Set (E6240L). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 10 or 14 cycles and library fragments of ~300 bp (insert plus adaptor and PCR primer sequences) were band isolated from an E-gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq2000 following the manufacturer's protocols.
GEO
GSM1847369