ChIP-Atlas: SRX957617

Antigen

Cell type Class: Blood
Cell type: Macrophages
MeSH Description: The relatively long-lived phagocytic cell of mammalian tissues that are derived from blood MONOCYTES. Main types are PERITONEAL MACROPHAGES; ALVEOLAR MACROPHAGES; HISTIOCYTES; KUPFFER CELLS of the liver; and OSTEOCLASTS. They may further differentiate within chronic inflammatory lesions to EPITHELIOID CELLS or may fuse to form FOREIGN BODY GIANT CELLS or LANGHANS GIANT CELLS. (from The Dictionary of Cell Biology, Lackie and Dow, 3rd ed.)

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: 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.