GSM9002360: ChIP_WT-SL_PolIIS5P; Mus musculus; ChIP-Seq
ChIP was performed as described previously (Nozawa et al., NCB, 2010) with a few modifications. Approximately 1×107 cells were prepared in 10 cm culture dishes and crosslinked with 5% formaldehyde (Thermo Fisher Scientific, 28908) for 5 min. Crosslinking was quenched with 200 mM glycine for 5 min and then cells were lysed with the lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 0.5% NP-40 or 0.5% IGEPAL CA-630, 0.1mM PMSF, 2 µg/ml leupeptin) for 7 min. Cells were collected in 1.5 ml tubes and centrifuged at 800g for 3 min. The supernatant was then discarded, and pellets were resuspended with the SDS lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 1% SDS, 0.1mM PMSF, 2 µg/ml leupeptin). Lysates were diluted with ChIP dilution buffer (50 mM Tris-HCl, 167 mM NaCl, 1.1% Triton X-100, 0.11% SDS, 0.1mM PMSF, 2 µg/ml leupeptin) and sonicated for 24 cycles (15 s ON/30 s OFF per cycle) using Bioruptor (Cosmo Bio, UCD-250). The sonicated chromatin was centrifuged at 16,000g for 15 min and the supernatant was collected. The chromatin was then snap-frozen in liquid nitrogen and stored at -80˚C until further use. 50 µl of Dynabeads M-280 Sheep anti-Mouse IgG (Thermo Fisher Scientific, 11202D) for mouse IgG or Dynabeads Protein A (Thermo Fisher Scientific, 10002D) for rabbit IgG were incubated with 1 µg of purified IgG or 10 µl of hybridoma culture supernatant at 4°C for 2 hours. Chromatin from 3–5×106 cells was incubated with the pre-incubated beads at 4˚C for 2 hours, followed by washing once with RIPA-150 buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 0.1 mM PMSF, 2 µg/ml leupeptin), once with RIPA-500 buffer (50 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 0.1 mM PMSF, 2 µg/ml leupeptin), and twice with TE buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1 mM PMSF, 2 µg/ml leupeptin). Reverse-crosslinking and elution were performed with ChIP direct elution buffer (10 mM Tris-HCl, 300 mM NaCl, 5 mM EDTA, 0.5% SDS, 0.1 mM PMSF, 2 µg/ml leupeptin) at 65°C overnight. The eluted chromatin was treated with 0.5 µg/ml RNase at 37°C for 30 minutes, followed by the addition of 50 µg/ml Proteinase K at 55°C for 1 hour. ChIPed or input DNA was purified using a QIAquick PCR Purification Kit (Qiagen, 28104). ChIP libraries were prepared with up to 20 ng of DNA using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L). Library quality control was performed using a Qubit 4 Fluorometer (Thermo Fisher Scientific, Q33238) and an Agilent 2100 Bioanalyzer (Agilent Technologies), followed by sequencing on an Illumina NovaSeq X Plus platform with a paired-end 150 bp system (Macrogen Japan Inc.).
GEO
GSM9002360_r1