title: GSM1944761: Rpc155_kcl; Mus musculus; ChIP-Seq
library_construction_protocol: Crosslinking buffer (0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA and 25 mM HEPES-KOH, pH 8.0) containing 1% formaldehyde was added to cultured cells for 10 min at room temperature. The cross-linking reaction was stopped by adding glycine to a final concentration of 125 mM. Cells were rinsed three times with ice-cold PBS containing protease inhibitor cocktail and 1mM PMSF), collected by scraping and centrifuged at 3000rpm at 4°C for 5 minutes. Cell pellets were transferred to 1.5 ml tubes and lysed with 20 cell pellet volumes (CPVs) of buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, pH 8.0, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100 and complete protease inhibitor cocktail) for 10 min at 4 °C. Nuclei were pelleted by centrifugation at 3000 rpm for 10 min at 4 °C,  incubated with 20 CPVs of buffer 2 (200 mM NaCl, 1 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, 10 mM Tris-HCl, pH 8.0, and complete protease inhibitor cocktail) for 10 min at RT and re-pelleted.  4 CPVs of buffer 3 (1 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, 10 mM Tris-HCl, pH 8.0, and complete protease inhibitor cocktail) were added to the nuclei, and sonication was carried out by applying 30 pulses, 30 seconds each, at 30 seconds intervals. Insoluble materials were removed by centrifugation at 14000 rpm for 10 min at 4 °C, the supernatant was transferred to a new tube and the final volume of the nuclear lysate was adjusted to 1 mL by adding buffer 3 supplemented with 0.3 M NaCl, 1% Triton X-100 and 0.1% deoxycholate. The lysates were pre-cleared with 70μL of Protein A-Sepharose beads (1 hour at 4°C) and subjected to immunoprecipitation. One tenth of the chromatin was saved for input control, while the remaining fraction was incubated with 3-5μg of antibody and rotated overnight at 4°C. Immune complexes were purified by incubating the lysates with 80μl of Protein A-Sepharose beads for 1 hour at 4°C. Beads were pelleted and washed twice with each of the following buffers: low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, and 150 mM NaCl), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris- HCl, pH 8.1, and 500 mM NaCl) and LiCl buffer (0.25 M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA and 10 mM Tris, pH 8.1). For each wash, the beads were incubated for 10 min at 4 °C while rotating. Beads were rinsed once with 1× TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) and the immunoprecipitated material was eluted by vortexing the beads twice for 15 min at RT in elution buffer (0.1 M NaHCO3 pH 8.0, 1% SDS). Crosslinking was reversed by adding 10μl 5M NaCl and incubating the samples at 65°C over night. DNA was purified using PCR purification columns (Qiagen) according to the manufacturer’s instructions. Libraries were prepared for sequencing using NEBNext ChIP-Seq Library Prep Master Mix (NEB) or Microplex Library preparation kit (Diagenode)
attributes.tag: GEO Accession
attributes.value: GSM1944761