title: GSM4433129: ChIP-seq_H2A.Z_DRB_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP was performed in ~10 million ES cells, per assay, as previously described (Mylonas & Tessarz, 2018). Briefly, cells were cross-linked with 1% formaldehyde for 10 min followed by by 5 min quenching with 125 mM glycine. After washing with PBS buffer, the cells were collected and lysed in cell lysis buffer (5 mM Tris, pH 8.0, 85 mM KCl, and 0.5% NP-40) with x1 Halt Protease Inhibitor cocktail (ThermoFisher 87786) and 1mM PMSF (Sigma 10837091001). Pellets were spun for 5 min at 6000 rpm at 4°C. Nuclei were lysed in nuclei lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris–HCl) and samples were sonicated for 12 min on a Covaris Sonicator. The samples were centrifuged for 20 min at 13,000 rpm at 4°C and the supernatant was diluted in IP buffer (0.01% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, and 167 mM NaCl), and the appropriate antibody (10 μg) was added and incubated overnight at 4°C with rotation. Antibodies used in this study; Anti-HA (Cell Signalling 3724), Anti-Spt5 (Santa Cruz, sc-133217), Anti-Dendra2 (OriGene TA180094). Two biological replicates were prepared for each condition using independent cell cultures and chromatin precipitations. The next day, 50 μl Protein G Dynabeads (Life Technologies #10009D) were added for 1 h at room temperature with rotation. Beads were washed once for 1 min with rotation with each of the following buffers: Low salt buffer (0.1% SDS, 1% Triton-X-100, 2mM EDTA, 20mM Tris pH 8.0, 150mM NaCl), High salt buffer (0.1% SDS, 1% Triton-X-100, 2mM EDTA, 20mM Tris pH 8.0, 500mM NaCl), LiCl buffer (0.25M LiCL, 1% NP-40, 1% dioxycholate, 10mM Tris pH 8.0, 1mM EDTA), and TE buffer (50mM Tris pH 8.0 , 10mM EDTA). DNA was eluted off the beads by rotation at room temperature for 30 min in 200 μL elution buffer (1% SDS, 0.1M NaHCO3). Cross-links were reversed at 65°C for 4h. RNA was degraded by the addition of 3 μL of 33 mg/mL RNase A (Sigma, R4642) and incubation at 37°C for 2 hours. Protein was degraded by the addition of 5 μL of 20 mg/mL proteinase K (Invitrogen, 25530049) and incubation at 50°C for 1 hour. A phenol:chloroform:isoamyl alcohol extraction was performed followed by an ethanol precipitation. The DNA was then resuspended in 20 μL H2O and used for either qPCR or sequencing. For ChIP-seq experiments, purified 10-20 ng of ChIP DNA was used to prepare Illumina multiplexed sequencing libraries. Libraries for Illumina sequencing were prepared following the NEBNext DNA Library Prep Master Mix kit (NEB E6040). Amplified libraries were size-selected using a 2% agarose gel to extract fragments between 200 and 600 bp.
attributes.tag: GEO Accession
attributes.value: GSM4433129