GSM2666938: BirA_control; Mus musculus; ChIP-Seq
Cells were cross-linked in 1% formaldehyde for 7 min at room temperature and the reaction was quenched for 5 minutes by adding glycine to final concentration of 125mM. Fixed cells were rinsed with PBS and collected in a tube after which the cells were rinsed with PBS twice. Cells were centrifuged and the pellets were used immediately for experiments or stored at -80°C. Cells were re-suspended in ChIP buffer (1% TritonX-100, 2mM EDTA, 20mM TrisCl, pH 8.1, 150mM NaCl, 0.1% SDS and protease inhibitor). Cells were then sonicated for 30 min (30 sec on / 1 min off) and centrifuged at maximum speed for 10 min. The supernatant was transferred to new tubes and pre-cleared with Protein A beads for 4 hours, rotating in 4°C. The samples were then centrifuged and the supernatant was incubated in 10μg streptavidin beads overnight (Roche). The beads were washed for 8 minutes, twice with 2% SDS, once with high salt buffer (0.1% Deoxycholate, 1% Triton X-100, 1mM EDTA, 50mM HEPES (pH 7.5), and 500mM NaCl), once with LiCl wash buffer (250mM LiCl, 0.5% NP40, 0.5% Deoxycholate, 1mM EDTA, and10mM TrisCl pH 8.1) and twice with TE buffer. Samples were eluted by incubating the beads in 150μl of SDS Elution buffer (1% SDS, 10mM EDTA and 50mM TrisCl pH 8.1) overnight in 65°C water bath, then for 30 min in 65°C water bath with 50ul of SDS Elution buffer. 200μl of TE buffer was added to the eluted samples and were treated with 1μg RNase A for 30 minutes in 37°C and 1μg Protease K for at least 2 hours in 37°C. ChIP-seq library prep kits (New England BioLabs) were used to generate ChIP-seq libraries. ChIP-seq libraries were sequenced using an Illumina HiSeq 2500 at the Genomic Sequencing and Analysis Facility (GSAF) of The University of Texas at Austin.
GEO
GSM2666938
GEO Accession
GSM2666938