GSM7623386: Cut&Tag_INTS5_SRCAP_degron_1h_rep2; Mus musculus; OTHER
DNA was purified through the Tris saturated equilibrium phenol (phenol: chloroform: isoamyl alcohol (25: 24: 1) (pH8.0) for ChIP-Seq, and gel extraction using DNA extract kit for Cut&Tag ChIP-seq, briefly, the pretreated cells were harvested by trypsinization following 1% formaldehyde (final concentration, wt/vol) crosslinked and stored at -80 °C for downstream experiments. For chromatin immunoprecipitation, 20 million crosslinked cells (5% HEK293T cell were added as spikein) were lysed gently for chromatin isolation with ice-cold NP-40 lysis buffer and glycerol / urea buffer, finally centrifugated at 12,000 rpm for 1 min at 4 °C, the pellet of chromatin were resuspended in 1 ml sonication buffer, 1000 U MNase (NEB, M0247) and 5 mM CaCl2 were added and incubated for 15 min at 37 °C with 750 rpm shaking. After quenched by 20 μl 0.5 M EDTA and 40 μl 0.5 M EGTA in ice bath, the chromatin was sonicated using Qsonica Q800R3 DNA Shearing Sonicator for 15 min with 60% AMPL. Soluble chromatin was isolated by centrifugation at 1,2000 rpm for 10 min at 4 °C, 20 μl supernatant were sampled as input, the rest supernatant was transferred into 2 new DNase free tube for immunoprecipitation. 1 μl GFP abs (ABcam, ab290), 1 μg RPB1 NTD abs (CST, 14958S), and equal isotype IgG control for each ChIP reaction, respectively. After incubation overnight at 4 °C with low-speed rotation, 30 μl Protein G magnetic beads were added into the mixture to capture for 4 h, then washed beads once with sonication buffer, twice with high-salt wash buffer, twice with LiCl wash buffer and finally three times with TE buffer. For ChIP-seq, beads were eluted and then decrosslinked with 4 μl proteinase K (Sigma, 20 mg/ml) at 65 °C overnight, DNA was purified through the Tris saturated equilibrium phenol (phenol: chloroform: isoamyl alcohol (25: 24: 1) (pH8.0), and the ChIP library was prepared following the instruction of VAHTS Universal Pro DNA Library Prep Kit for Illumina (Vazyme, ND608-01); Cut&Tag was performed according to the manufacture of Hyperactive Universal Cut&Tag Assay Kit for Illumina (Vazyme, TD903-01), with 5% spikein 293T cells ChIP-Seq, Cut&Tag