title: GSM2111317: Ly6Chi_H3K4me2; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP assays for histone modifications were performed as previously described (Gilfillan et al., 2012). For each ChIP assay 500,000 FACS isolated cells were immediately washed with PBS and resuspended in MNase digestion buffer (50mM Tris pH 8.0, 1mM CaCl2, 0.2% Triton X-100). Cells were then digested in to mononucleosomal fragments using micrococcal nuclease (MNase, Affymetrix, CA). Enzymatic digestion was quenched by addition of 1/10 volume stop buffer (110mM Tris pH 8.0, 55mM EDTA), samples briefly sonicated using a Bioruptor (Diagenode, Belgium) and then adjusted to RIPA buffer conditions by adding an equal volume of 2x RIPA buffer (280mM NaCl, 1.8% Triton x100, 0.2% SDS, 0.2% Sodium Deoxycholate, 5mM EGTA). Immunoprecipitations were performed in a final volume of 100 μl. 2 μg of anti-H3K4me2 (Millipore 07-030) or 2μg of anti-H3K27ac (Diagenode C15410196) were added and incubated overnight with rotation at 4 °C. Antibody-antigen-DNA complexes were recovered by incubating for 2 hours with 10 μl Protein A Dynabeads previously washed in RIPA buffer (10mM Tris pH 8.0, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.2% SDS, 0.2% Sodium Deoxycholate). Complexes were then washed 5x in ice cold RIPA buffer and 1x in LiCl wash buffer (10mM Tris pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% Igepal CA-630, 0.5% Sodium deoxycholate,), each wash was performed in 200 μl at 4 °C for 5 minutes with rotation. All above buffers were supplemented with 1x Protease inhibitor Cocktail, 1mM PMSF and 5mM Sodium Butyrate (Sigma). Beads were subject to a final wash in 200 μl ice cold TE buffer (Invitrogen) without protease inhibitors and eluted in 100 μl 1% SDS-TE buffer at 37 °C for 20 minutes. Finally, protein was treated with 2 μl proteinase K (Ambion) for 1h at 55 °C and DNA purified using a ChIP Clean & Concentrate column (Zymo, Irvine, CA) eluting in 30 μl final volume.      ChIP for PU.1 and C/EBPb were performed as previously described (Gosselin et al., 2014). 500,000 sorted cells were washed in PBS and immediately fixated for 9 minutes at room temperature in 1% methanol free formaldehyde in PBS (ThermoFisher Scientific). Fixation was quenched by addition of 1/20 volume 2.625M glycine solution and cells were washed twice with PBS. Cell pellets were then snap frozen in a dry ice/methanol bath and stored at -80oC until needed. Nuclei were enriched by resuspending cell pellets in 10mM HEPES pH 7.9, 85mM KCl, 1mM EDTA, 0.5% Igepal CA-630 and incubating on ice for 10 minutes. Nuclei were harvested by spinning at 3000 g for 10 minutes, and lysed in 130 μl lysis buffer (10mM Tris pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-lauroylsarcosine). DNA was transferred in to Covaris micro tubes (Covaris, MA) sheared into 150-600bp fragments using a Covaris E220 (14 mins, duty cycle 3%, 100 cycles per burst). Final volume was adjusted to 200 μl and 22 μl 10% Triton X-100 added, cell debris was then cleared by spinning at maximum speed for 5 minutes. 20 μl of protein A dynabeads pre-conjugated to 3ug anti PU.1 or C/EBPb (sc-352x and sc-150x respectively, Santa Cruz Biotechnology, CA) were added to each chromatin preparation and incubated with rotation for 2h at 4 °C. Antibody-antigen-DNA complexes were then washed in 3x WBI (20mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2mM EDTA, ), 3x LiCl WB (10mM Tris Ph 7.4, 250mM LiCl,, 1% Triton X-100, 0.7% Sodium Deoxycholate, 1% Igepal CA-630) and 1x TET (TE, 0.2% Tween-20) buffer. All wassh volumes were 200 μl. DNA was eluted in 1% SDS-TE for 30 minutes at 37 °C, NaCl was added to a final concentration of 300mM and corsslinking reversed overnight at 65 °C. Finally, protein was treated with 2 μl proteinase K (Ambion) for 1h at 55 °C and DNA purified using a ChIP Clean & Concentrate column (Zymo, Irvine, CA) eluting in 30 μl final volume. ChIP-Seq libraries were prepared using an initial 0.3 - 5 ng DNA using the ThruPlex-FD kit (Rubicon Genomics, MI) in accordance with the manufacturer's guidelines. RNA-Seq libraries were prepared using the Tru-Seq v2 library preparation kit (Illumina, La Jolla, CA) in accordance with the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM2111317