GSM1692866: MES_Elk4; Mus musculus; ChIP-Seq
Cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature. The reaction was quenched with 0.125M glycine, incubating at room temperature for 5 minutes. Cells were immediately harvested and washed in cold 1x PBS. Cells were harvested and resuspended in 1.5 x pellet volume of cell lysis buffer (10mM Tris pH 8.0, 10mM NaCl and 0.2% NP40) containing protease inhibitors (leupeptin, NaBu and PMSF), the cells were homogenised and incubated on ice for 10 minutes. The following conditions are for 108 cells and were scaled down for lower cell numbers.  nuclei were harvested at 600 x g for 5 minutes at 4oC and resuspended in 1 ml of nuclei lysis buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS) containing protease inhibitors (leupeptin, NaBu and PMSF), the cells were homogensised and incubated on ice for 10 minutes. An equal volume of IP dilution buffer (20mM Tris pH 8.0, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS) containing protease inhibitors (leupeptin, NaBu and PMSF) was added. Cells were sonicated on ice-water or chilled water (Biorupter or Biorupter-plus, Diagenode) for 5 cycles (30s on, 30s off). The chromatin solution was centrifuged for 10 minutes at 3220 x g. After transferring the chromatin solution to a clean falcon tube, a further 3 mls of IP buffer was added. The chromatin solution was pre-cleared by the addition of 50μl of IgG (2 μg/μl, raised in the same species as the experimental antibody) and incubated at 4oC for 1 hour, then 200μl of Protein G sepharose beads (1:1 slurry in IP dilution buffer) were added to the chromatin solution and further incubated at 4oC for 2 hours. The beads/IgG were collected by centrifugation at 1791 x g for 2 minutes. The chromatin was transferred to 1.5 ml tubes, an input sample was removed and antibodies/IgG were added then incubated overnight at 4oC with rotation. In the case of low cell number samples (HEs) 1 μl mouse mRNA (diluted 1:5, cat# 338114, Qiagen) and 4 μl Recombinant Histone 2B (M2505S; New England Biolabs) was added prior to the antibody. 60μl of protein G agarose beads (1:1 slurry in IP dilution buffer) were added and incubated with the samples for 2 hours. The beads were harvested at 5400 x g for 2 minutes and washed twice with low salt buffer (20mM Tris pH 8.0, 2mM EDTA, 50mM NaCl, 1% Triton X-100, 0.1% SDS), then once with LiCl buffer (10mM Tris pH 8.0, 1mM EDTA, 0.25M LiCl, 1% NP40, 1% Sodium deoxycholate monohydrate) and twice with 1x TE pH 8.0. The complexes were eluted twice from the beads by adding 150μl elution buffer (100mM NaHCO3, 1% SDS). To reverse the cross-linking 0.3M NaCl was added to all the IP samples and input, RNase was added and the samples were incubated at 65oC overnight. The samples were then treated with Proteinase K for 2 hours at 45oC. DNA was purified using Qiagen PCR clean up columns. The libraries were prepared according to Illumina TruSeq DNA Sample Prep Kit
GEO Accession
GSM1692866