GSM5856484: H3K27ac Lung 2; Mus musculus; ChIP-Seq
Flow-sorted endothelial cells were fixed using 1% formaldehyde. 200,000 cells were lysed and frozen in 100ul SDS lysis buffer (50mM Tris/HCl, 0.5% SDS, and 10mM EDTA) supplemented with 1x cOmplete EDTA-free protease inhibitor (Roche) (n=3 mice of each genotype, n=6 for heart H3K27ac). For ChIP-seq, anti-FLAG (Cell Signaling Technologies, #8146), anti-Pol II (Sigma, 05-623), anti-p300 (Cell Signaling Technologies, 54062), or anti-H3K27ac (Abcam, ab4729) antibodies were added to Protein A/G-coupled Dynabeads (Bimake, B23202) in PBS with 0.5% BSA and incubated with rotation for 4hr at 4 degrees. For 100,000-150,000 cells, 25 ul of beads were incubated with 5ug antibody per ChIP. Aliquots were diluted with SDS lysis buffer and 100 ul containing the appropriate number of cells were processed. Cells were sonicated for 12 cycles of 30s on/30s off on high power using a Bioruptor Pico (Diagenode). To neutralize the SDS, Triton-X-100 was added to a final concentration of 1% along with 50x cOmplete protease inhibitor (final concentration: 1x). Samples were incubated at room temperature for 10min and 5% aliquots were saved for preparation of input controls. Antibody-coated Dynabeads were washed with PBS and mixed with cell lysate and incubated overnight at 4 degrees with rotation. Immunoprecipitated chromatin was washed with 150ul low-salt buffer (50mM Tris/HCl, 150mM NaCl, 0.1% SDS, 0.1% NaDOC, 1% Triton-X-100, and 1mM EDTA), high-salt buffer (500mM NaCl), and LiCl buffer (10mM Tris/HCl, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% NaDOC, and 1mM EDTA), followed by two washes with TE buffer and two washes with ice cold Tris/HCl (pH 8, 10mM). For tagmentation, bead-bound chromatin was resuspended in 30ul of tagmentation buffer, 1ul of transposase (Nextera, Illumina) was added and samples were incubated at 37 degrees for 10min, followed by two washses with low-salt buffer. For standard reverse crosslinking, chromatin complexes were diluted with 200ul ChIP elution buffer (10mM Tris/HCl, 0.5% SDS, 300mM NaCL, and 5mM EDTA) and 2ul of 20ug/ml proteinase K (Thermo Scientific). Samples were vortexed and incubated overnight at 65 degrees. After reverse crosslinking, 1 ul 20ug/ml RNase (Sigma) was added and incubated at 37 degrees for 30min. DNA purification was carried out using Qiagen MinElute PCR Purification Kit. 15ul of PCR master mix and 5ul of primer mix (Nextera, Illumina) were added to 20ul of eluted DNA and libraries were amplified. After PCR amplification, library cleanup was done using Agencourt AmPureXP eads (Beckman Coulter) at a ratio of 1:1. DNA concentrations in purified samples were measured using the Qubit dsRNA HS Kit (Invitrogen). Libraries were pooled and single-end sequenced (50 cycles) using the NextSeq500 platform (Illumina). Analysis was performed as described in the ATAC-seq pipeline