GSM5740110: Sample_p53_ChiPseq_TPC1 - DMSO - rep2; Homo sapiens; ChIP-Seq
GSM5740110
Sub-confluent cultures of TPC1 and K1 cells were treated for 24 hours with indicated compounds. After the treatment period, cultivation media was replaced with crosslinking solution (1% formaldehyde in PBS) and plates were incubated for 15 minutes at room temperature. Next, formaldehyde was quenched with glycine (0.125 mM final) for 5 minutes and cells were washed twice with ice-cold PBS. Crosslinked cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris pH 8.5 mM EDTA, 1% IGEPAL 630 (NP-40 substituent), 0.5% sodium deoxycholate, 0.1% SDS and protease/phosphatase inhibitors) and sonicated to generate 200-300 bp fragments of DNA (Qsonica Q800R, 70% amplitude, 30 sec on/30 sec off cycle, 20 cycles for TPC1 lysates and 25 cycles for K1 lysates). Next, samples were centrifugated at 20 000 g for 20 min at °C, protein concentration in collected supernatants was measured using a BCA Protein Assay Kit and all samples were diluted to final protein concentration of 1 mg/ml. Lysates were pre-cleared with 15 l of Dynabeads M-280 (sheep anti-mouse IgG, Thermo Fisher Scientific) and immunoprecipitated overnight with 5 l/sample of anti-p53 antibody (DO-1, EMD Millipore) and 30 l of Dynabeads at 4C. In total, 4 (TPC1) or 5 (K1) lysate aliquots per sample were used in immunoprecipitation reactions. Next day beads were washed (5 minutes each washing step) twice with RIPA, four times with IP wash buffer (500 mM LiCl, 100 mM Tris pH 8.5, 1% IGEPAL, 1% sodium deoxycholate), again twice with RIPA and twice briefly with TE (10 mM Tris pH 8, 1 mM EDTA). Washed beads were resuspended in 100 l of TE and 200 l of elution buffer (70 mM Tris pH 8, 1 mM EDTA and 1.5% SDS) and incubated at 65C for 10 minutes. After adding NaCl to final concentration of 200 mM, eluted immunocomplexes were incubated at 65C for 5 hours to reverse formaldehyde crosslinks. Remaining protein was digested by proteinase K (20 g/sample, 45C for 30 minutes). DNA was recovered by one phenol/chloroform and one chloroform extraction followed by ethanol precipitation and resuspension in 50 l of TE. Input DNA was extracted from reverse cross-linked lysates using the same extraction protocol as for sample DNA. NEBNext Ultra II DNA sequencing library preparation kit