Name: GSM7372132
Instrument: NextSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Cells were crosslinked in 1% formaldehyde in DMEM for 10 min at RT. Next, glycine was added to a final concentration of 0.125 M and cells were incubated for 5 min at RT to stop the fixation. After two washes with ice-cold PBS, cells were harvested by gently scrapping on ice and centrifuged at 3,000 x g for 5 min. Pellets were stored at 80ºC until use. Chromatin preparation: cells were lysed in hypotonic lysis buffer (5mM Pipes pH 8; 85mM KCl; 0.5% NP40; plus protease inhibitors). Nuclei were recovered by centrifugation, lysed in nuclear lysis buffer (50mM Tris HCl pH 8; 1% SDS; 10mM EDTA plus protease inhibitors) and sonicated with a Bioruptor Pico, Diagenode (20 cycles 30” ON, 30” OFF) to yield chromatin fragments of 150-300 bp. For ChIP experiments, chromatin (25 µg DNA) was diluted 1/10 in ChIP Buffer (16.7mM Tris HCl pH 8; 167mM NaCl; 1.2mM EDTA; 1.1% Triton x100 plus protease inhibitors) and mix with 2% Drosophila Spike-in chromatin. Aliquots were removed as input material (1%). ChIP samples were incubated with the specific antibody (5 µg) plus Spike-in antibody (1µg) overnight 4ºC, and immunoprecipitated with Protein A agarose beads (Diagenode) during 2 h at 4°C. Beads were washed 3 times with low-salt buffer (20mM Tris HCl pH8; 150 mM NaCl; 2mM EDTA; 0.1% SDS; 1% Triton X-100) and once with high-salt buffer (20mM Tris HCl pH8; 400 mM NaCl; 2mM EDTA; 1% Triton X-100; 0.1% SDS). ChIPed material was eluted from the beads in Elution buffer (1% SDS, 100 mM NaHCO3) at 65°C in a shaker (1000 rpm) for 1 h. Eluted material and inputs were de-crosslinked overnight at 65°C, treated with Proteinase K and the DNA purified using the QIAquick PCR purification kit (Qiagen). The following antibodies were used in the ChIP experiments: H3K4me3 (Diagenode, C15410003); H3K27Ac (Millipore, #07–360); Drosophila H2Av (Active Motif #61686). Libraries were prepared according to Illumina instructions.