GSM3891952: input DNA; Mus musculus; ChIP-Seq
Nuclei were isolated using lysis buffer 1 [5 mM Pipes pH 8, 85Mm KCl, 0.5% NP40, complete protease inhibitor cocktail (Roche)] and were lysed using lysis buffer 2 [1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, complete protease inhibitor cocktail (Roche)]. Chromatin was sheared into an average size of 500 pb by 8 30s pulses (30s pause between pulses) for P19 cells at 4º C in the waterbath sonicator Bioruptor (Diagenode, Liège, Belgium). 30 µg of chromatin from P19 cells, were incubated overnight at 4º C in rotation diluted 1:10 in IP buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl] and with 3 µg of anti-NIPBL (A301-779A, Bethyl Laboratories, Inc.). IPs were incubated for 2h at 4º C in rotation with protein A or G Dynabeads (Invitrogen) for rabbit or mouse antibodies, respectively, and then washed with wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), wash buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), wash buffer 3 (0.25 M LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), and twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The complexes were eluted from the beads with elution buffer (1% SDS in TE buffer) by incubating twice 10 min at 65º C. The eluates and the inputs (3 or 1 µg of chromatin from P19 cells or human fibroblasts, respectively) were incubated overnight at 65º C for de-crosslinking. All the samples were treated with proteinase K (Roche) for 1h at 37ºC and purified using the ChIP DNA Clean and & Concentrator kit (Zymo Research, Irvine, CA, USA). Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit