title: GSM2427792: Neuro2a_ChIPseq_input; Mus musculus; ChIP-Seq
library_construction_protocol: Neuro 2a cells were grown to confluency on 150 mm culture dishes. Cells were crosslinked directly on the dishes for 10 min at room temperature with 1% formaldehyde, followed by quenching with 0.125M glycine for 5 min. Cells were scraped, pelleted, and lysed in cell lysis buffer (50 mM HEPES (pH 7.5) 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.25% Triton X-100, 0.5% NP-40, 10% glycerol) for 10 min on ice. Nuclei were collected and lysed in 10 mM Tris pH 8.0, 1% SDS, 1 mM EDTA and 1 mM EGTA. The SDS concentration was diluted to 0.625% and DNA shearing was performed on a Bioruptor instrument (Diagenode; 5 ten-minute cycles, 30 sec on, 30 sec off). Buffer conditions were adjusted to contain 150 mM NaCl, 0.1% SDS, and 0.5% Triton X-100 and chromatin was precleared with Protein A Dynabeads (Invitrogen) for 2 h and an aliquot was saved as Input. Immunoprecipitation was performed using 32 ul Protein A Dynabeads and 5ul of H3K4me3 antibody (Millipore; 07-473) or 10ug H3K27ac antibody (Abcam; ab4729). Beads were washed twice with low-salt buffer (150 mM NaCl, 50 mM HEPES pH 7.5, 0.1% DOC, 1% Triton X-100, and 1 mM EDTA), once with high-salt buffer (500 mM NaCl, 0.1% DOC, 50 mM HEPES pH 7.5, 1% Triton X-100, and 1 mM EDTA), once with LiCl buffer (10 mM Tris-HCl pH 8.1, 250 mM LiCl, 0.5% NP-40, 0.5% DOC, 1 mM EDTA), and twice with TE. Chromatin was eluted with elution buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 1% SDS, wt/vol) and reverse crosslinked overnight at 65º C, followed by treatment with RNase A for 30 min at 42º C and proteinase K for 3 hr at 55º C. DNA was extracted twice with phenol/chloroform, once with chloroform, and ethanol precipitated. 10 ng of ChIP DNA was used for library preparation. Libraries were prepared for sequencing using standard Illumina protocols.
attributes.tag: GEO Accession
attributes.value: GSM2427792