GSM4230013: TGFB_pSmad3-ChIPseq_rep2; Mus musculus; ChIP-Seq
50 million cells per replicate were cross-linked with 1% formaldehyde for 10 min, then lysed in modified RIPA buffer. Cellular extracts were sonicated using a Covaris B208 ultrasonicator to produce chromatin fragments 100–400 bp in length. Cleared extract was incubated with 10 μg of antibody against phosphoSmad2/3 (abcam ab52903) or nonspecific rabbit IgG control antibody (Cell Signaling) overnight and precipitated using magnetic Protein A Dynabeads (Invitrogen). Beads were washed with ChIP wash buffer and protein–DNA complexes were eluted with 1% SDS, 50 mM NaCOH3, then cross-links were reversed by incubation with 0.2M NaCl at 67 °C overnight. DNA was isolated using Qiagen MinElute columns following the manufacturer's instructions, and used for ChIP-seq library prep. ChIP–seq libraries were generated using the standard protocol for the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs)
GEO Accession
GSM4230013