GSM8766946: INTS3_NELFC-KD_R2; Homo sapiens; ChIP-Seq
Cells were cultivated on 150 mm dishes until 50% confluency, fixed by addition of 1% formaldehyde (Sigma) for 15 min at 37°C and quenched by 125mM glycine for 10 min at 37°C. The cells were collected by scraping, washed 3 times with cold PBS, resuspended in 1.5 mL L1 buffer (50 mM Tris pH 8.0; 2 mM EDTA pH 8.0; 0,1% NP40; 10% glycerol; protease inhibitors (Nacalai Tesque)) per 107 cells, and lysed on ice for 5 min. The nuclei were collected by centrifugation at 800g for 5m at 4°C and lysed in 1.5 mL of L2 buffer (0,2% SDS; 10 mM EDTA; 50 mM Tris pH 8.0; protease inhibitors (Nacalai Tesque)). The suspension was sonicated in 15 mL conical tubes in a cooled Bioruptor 2 (Diagenode) for 15 min at low settings. Sonicated chromatin was centrifuged for 10 min at 1,4000 rpm and collected the supernatant. The shared chromatin was diluted 10x in the dilution buffer.  For calibration, 4 ng of spike-in chromatin (Active Motif) was added to each IP sample. 60 µg of Protein A Dynabeads (Thermo Fisher Scientific) for each sample were pre-washed twice with dilution buffer (0,5% NP40; 200 mM NaCl; 50 mM Tris pH 8.0; protease inhibitors). The chromatin was incubated with pre-washed beads for 1h at 4°C to clear unspecific binding proteins to the beads. 3μg of the pre-cleared chromatin was incubated with 6 μg of α-CSTF64 (A301-092A, Bethyl) or α-INTS3 (16620-1-AP, Proteintech) and 4.4 μg of spike-in specific antibody (Active Motif), O/N at 4°C on rotator. Next, 60 μL pre-washed beads were added to the chromatin+antibody sample and incubated for 1-2hr at 4°C on rotator. After the incubation, the beads were washed 2 times with buffer A (0.05% SDS; 0.5% TritonX; 2 mM EDTA; 150 mM NaCl; 20 mM Tris pH 8.0), 3 times with buffer B (0.02% SDS; 0.5% NP40; 2 mM EDTA; 250 mM NaCl; 20 mM Tris pH 8.0), 2 times with buffer C (0.05% DOC; 0.5% NP40; 2 mM EDTA; 500 mM NaCl; 20 mM Tris pH 8.0) and finally once again with buffer D (1 mM EDTA; 10 mM Tris pH 8.0). The immunoprecipitated DNA fragments were eluted from the beads with 100ul elution buffer (1% SDS, 100mM NaHCO3) for 15 min at 30°C, and repeated this step twice. After elution, for de-crosslinking, the DNA fragments were incubated for 4h at 65°C in the presence of 10µg RNase A (EN0531, Thermo Fisher Scientific) on thermo mixer (eppendorf). The immunoprecipitated DNA was then purified with the DNA Clean & Concentrator Kit (ZYMO) according to manufacturer's protocol and used for library preparation. NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) was used to prepare libraries for sequencing, following manufacturer's instructions.
GEO
GSM8766946_r1