GSM2344454: RNApolII_WT_LPS_UT; Mus musculus; ChIP-Seq
Roughly 80 x 10^6 (for Fli1, Elf1 and Pol II ChIP-seq), 40 x 10^6 (for GABP-α, Elf4 ChIP-seq) or 5 x 10^6 (for PU.1 and histone modifications ChIP-seq) cells were fixed in 1% formaldehyde for 10min and were lysed with RIPA buffer, after chromatin shearing by sonication. Lysate was immunoprecipitated with 5-10 μg of antibody. Antibodies were pre-bound overnight to 50-100 μl of G protein-coupled paramagnetic beads in PBS/BSA 0.5%. Beads were then added to lysates (the preclearing step was omitted) and incubation was let to proceed overnight. Beads were washed 6 times in a modified RIPA buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-deoxycholate) and once in TE containing 50mM NaCl. DNA was eluted in TE containing 2% SDS and crosslinks reversed by incubation overnight at 65ºC. DNA was then purified by Qiaquick columns (Qiagen) and quantified using PicoGreen (Invitrogen). ChIP DNA libraries were prepared for HiSeq 2000 Illumina sequencing using a standard protocol (Garber et al., 2012) with slight modifications (Ostuni et al., 2013).