title: GSM4504373: ChIP-Suz12_DMSO_1 [RAb5-2]; Mus musculus; ChIP-Seq
library_construction_protocol: Growing embryoid bodies (EBs) were transferred to a gelatin-coated plate on day 4 and maintained for 24 hours more. Total RNA was obtained using an RNA-zol extraction kit from Zymo Research with DNase treatment. For ChIP experiments, EBs were cross-linked with 1% PFA for 10 minutes, quenched with 0.125 M glycine, and chromatin was extracted and sonicated to obtain 200-500 bp fragments. 5 million cells were crosslinked with 1% formaldehyde in 1x PBS for 10 min and lysed in Farnham lysis buffer (5 mM PIPES pH 8.0; 85 mM KCl; 0.5% NP-40) passing the lysate through a 19-gauge needle 20 times. Nuclei were collected at 400 rcf for 5 min and resuspended in RIPA buffer (1x PBS; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS). Then, they were sonicated at 40% amplitude for 20 min (Qsonica, CT). Chromatin shearing between 150 and 500 pb was confirmed by 1% agarose gel electrophoresis. 25 ug of sample (A260 quantification) were combined with 5 ug of a Rabbit anti Suz12 (#39057, Active Motif, CA) or normal rabbit IgG bound to Dynabeads Protein A. Bead-antibody complexes were washed five times with IP wash buffer (100 mM Tris pH 7.5; 500 mM LiCl; 1% NP-40; 1% sodium deoxycholate), once with 1x TE, and DNA-proteins were eluted with 1% SDS; 0.1 M NaHCO3 for 1 h at 65°C. Crosslinking was reversed incubating the samples ON at 65°C. The next day, DNA was purified using the QIAquick PCR purification kit (Qiagen, Germany) and quantified for library preparation using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher). Total RNA isolated as described above and ChIP samples for sequencing were prepared using NEBNext library preparation kits from New England Biolabs. Poly(A) RNAs were isolated from RNA samples using the Poly(A) mRNA Magnetic Isolation Module (NEB). The first cDNA strand was obtained using Superscript III in presence of Actinomycin D, and the second cDNA strand was obtained using the Ultra Directional RNA Second Strand Synthesis module (NEB). From this point, both RNA- and ChIP-derived samples followed the same protocol: double stranded DNA was end-repaired, dA-tailed, adaptor-ligated and size-selected using the ChIP-seq library prep master mix set for Illumina (NEB). Size of the libraries was confirmed using high sensitivity DNA reagents for Bioanalyzer (Agilent, CA) and sample concentration was determined using the KAPA library quantification kit (Kapa Biosystems, MA). Libraries were sequenced in Illumina HiSeq sequencer.
attributes.tag: GEO Accession
attributes.value: GSM4504373