GSM5350985: H3K9ac ChIP-seq WT; Mus musculus; ChIP-Seq
Mouse ES E14 and Drosophila S2 cells were washed with 1x PBS, fixed at room temperature with 1% paraformaldehyde in 1x PBS for 10 minutes. Fixation was stopped by adding glycine to a final concentration of 125mM for 10 minutes at room temperature. Cells were washed twice with ice-cold 1x PBS and collected by scrapping. Cells were centrifuged at 2000g for 5 minutes at 4°C and washed once with ice-cold 1x PBS. Cells were lysed in L1 buffer (50 mM Tris-HCl ph 8.0, 2 mM EDTA pH 8.0, 0.1% NP40, 10% glycerol, 5 mM sodium butyrate and protease inhibitory cocktail) and incubated on ice for 10 minutes before centrifugation at 2000g for 10 minutes at 4°C. Nuclei were resuspended in L2 buffer (0.5% SDS, 10 mM EDTA ph 8.0, 50 mM Tris-HCl pH 8.0, 5 mM sodium butyrate and protease inhibitory cocktail) and sonicated using a Covaris E210 sonicator to on average 300 bp fragments. Protein-A sepharose beads were washed twice with TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) and blocked for three hours with 1ug/ul denatured yeast tRNA and 1,5% fish-skin gelatine. Beads were washed twice with TE buffer and stored at 4°C. For ChIP, 49 µg of mouse chromatin was spiked with 1 µg of fly chromatin. The chromatin mix was diluted with chromatin dilution buffer (0.01% SDS, 1.1% Triton-X 100, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl pH 8.0, 5 mM sodium butyrate and protease inhibitory cocktail) to a volume of 800 µl and incubated with 30 µl of blocked bead slurry for 1 hour at 4°C for preclearing. Two µg of anti-H3K9ac antibody (abcam, cat # ab4441) was added to precleared chromatin and incubated overnight at 4°C. 50 µl of bead slurry was added to samples and incubated for two hours at 4°C with overhead shaking. After centrifugation for 2 minutes at 1000g, beads were washed two times with Low salt washing buffer (0.1% SDS, 1% Triton-X 100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris HCl pH 8.0, 5 mM sodium butyrate and protease inhibitory cocktail) for 10 minutes, two times with High salt washing buffer (0.1% SDS, 1% Triton-X 100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris HCl pH 8.0, 5 mM sodium butyrate and protease inhibitory cocktail) for 10 minutes, two times with LiCl washing buffer (250 mM LiCl, 1% NP40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0, 5 mM sodium butyrate and protease inhibitory cocktail) for 10 minutes and two times with TE buffer for 10 minutes. Bound fragments were eluted using with freshly prepared elution buffer (1% SDS and 0.1 M sodium bicarbonate) at room temperature. Chromatin was reverse crosslinked by addition of 0.2M NaCl and 50 µg/ml RNase A. Samples were incubated at 37°C for one hour. 20 µg of Proteinase K, 60 mM Tris ph 7.9, 20 mM EDTA was added and samples were incubated at 65°C overnight in a thermomixer. DNA was isolated by Phenol-chloroform purification and resuspended in water. For library preparation, ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter, Cat#A63882) and quantified with the Qubit (Invitrogen). ChIP-seq libraries were prepared from 7 to 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (Diagenode, Cat# C05010014), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5' ends were ligated to the 5' end of the genomic DNA, leaving a nick at the 3' end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3' ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7 cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads to remove unincorporated primers and other reagents. The libraries were sequenced on Illumina Hiseq 4000 sequencer as single-Read 50 base reads following Illumina's instructions.