GSM1634816: wdr82sh_sh1137_H3K4me3_LPS_4h; Mus musculus; ChIP-Seq
Roughly 10-20 x 10^6 (for histone modifications) or 100-150 x 10^6 (for Pol II) fixed (1% formaldehyde for 10 min) macrophages were lysed with RIPA buffer and, after chromatin shearing by sonication.  Antibodies were pre-bound overnight to 100 ul of protein G-coupled paramagnetic beads (Dynabeads) in PBS/BSA 0.5%. Beads were then added to lysates and immunoprecipitation was let to proceed overnight. For Ser2-phospho RNA Pol II the ChIP lysates were incubated overnight with the primary antibody and then combined with 100 μl of beads that had previously been coupled overnight with 5 μg of rabbit anti-rat IgG (Jackson ImmunoResearch, #312-005-003). Beads were washed 6 times in a modified RIPA buffer (50mM Hepes pH 7.6, 500 mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-deoxycholate) and once in TE containing 50 mM 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). Library preparation for Illumina sequencing (HiSeq 2000) was carried out using a described protocol (Garber et al., 2012) with slight modifications (Ostuni et al., 2013). The purified DNA libraries were quantified both with a 2100 Bioanalyzer (Agilent Technologies) and Qubit (LifeTechnologies) and diluted to a working concentration of 10 nM.
GEO Accession
GSM1634816