GSM3964086: ChIP-Seq with no antibody in SCP3 positive STRA8 positive nuclei; Mus musculus; ChIP-Seq
Chromatin extracted for ChIP-Seq was either sheared by sonication or Micrococcal nuclease (MNase) digestion. Chromatin for all H3K4me3 ChIP-Seq for studying histone dynamics in stage-specific populations was sheared by sonication, whereas ChIP-Seq for identifying histone marks at hotspots were performed on MNase-digested chromatin. Experimental procedures are listed below in detail.     Shearing chromatin by sonication:  Frozen nuclei pellets were thawed at RT for 10 min. Nuclei were lysed with Lysis buffer 1 (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8) and incubated at RT for 10 min. Nuclei pellets were subsequently washed with Lysis buffer 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8) and lysed with RIPA buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl plus protease inhibitor). Chromatin was sheared into ~500−1000 bp fragments by sonication using Bioruptor (diagenode). Chromatin concentration was measured using a Qubit dsDNA HS Assay Kit (Thermo Fisher).    MNase digestion:  Frozen nuclei pellets were thawed at RT for 10 min. Nuclei pellets were resuspended with MNase buffer (50 mM Tris-HCl pH 8, 1 mM CaCl2, 4 mM MgCl2, 4% NP-40 plus protease inhibitor). MNase digestion was performed in a concentration of 3U MNase (USB, Affymetrix) per one million nuclei at 37°C for 5 min. The reaction was stopped by adding a final concentration of 10 mM EDTA and incubated at 4°C for 5 min. Chromatin was pelleted and resuspended in RIPA buffer. Chromatin concentration was measured. For stage-specific H3K4me3-ChIP-Seq, modified mononucleosomes (SNAP-ChIP K-MetStat Panel, EpiCypher) were added at a spike-in volume of 1 μl per ~1.5 μg chromatin DNA. Chromatin was immunoprecipitated with 0.8–5 μg antibodies (or 5 μl unpurified serum) in 0.5–1 ml RIPA buffer at 4°C overnight (see Supplementary Table 1 for details). The immuno-complexes were captured using 20–75 μl Dynabeads Protein G (30 mg/ml, Novex) at 4°C for 2 hr. The beads were washed with buffers previously described27. ChIPed DNA was eluted using a IPure kit v2 (diagenode). ChIP-seq libraries were constructed with a KAPA Hyper Prep Kit (Kapa Biosystems) following steps for generating 1 μg of library DNA. DNA libraries were cleaned up with an Agencourt AMPure XP system (Beckman Coulter). The DNA concentration and fragment size of these libraries were measured with a Qubit dsDNA HS Assay Kit (Thermo Fisher) and an Agilent High Sensitivity DNA Kit (Agilent), respectively.