GSM8155899: H3K27ac_FL_Shox2trac_E115_MAINTAIN_rep1; Mus musculus; ChIP-Seq
After sorting, cells were centrifuged for 5' at 400g at 4°C and supernatant was discarded. Cells for ChIP-seq were resuspended in 10% FCS/PBS and fixed in 1% PFA at room temperature. The fixation was blocked by the addition of 1M glycine, cells were isolated by centrifugation (1000g, at 4°C for 8'), resuspended in cold lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 5 mM MgCl2, 1mM EGTA, Protease Inhibitor (Roche, 04693159001)) and incubated on ice for 10' to isolate the cell nuclei. The nuclei were isolated by centrifugation (1000g, at 4°C for 3'), washed in cold 1X PBS, centrifuged again (1000g, at 4°C for 1') and stored frozen at -80°C after removal of the PBS supernatant. 500000 fixed nuclei were sonicated to a 200-500bp length with the Bioruptor Pico sonicator (Diagenode). H3K27Ac ChIP was (Diagenode C15410174) was performed as previously described (Lee et al. 2006; Rouco et al. 2021) with the addition of 5mM of Na-Butyrate to all buffers. Libraries were then prepared following the Illumina ChIP TruSeq protocol and sequenced as 50bp single-end reads on an Illumina NovaSeq 6000. Libraries were prepared starting with below <10ng quantities of ChIP-enriched DNA as starting material and processed with the Illumina TruSeq ChIP kit according to manufacturer specifications. Libraries were validated on a Tapestation 2200 (Agilent) and a Qubit fluorimeter (Invitrogen – Thermofisher Scientific). Libraries were pooled at 2 nM and loaded for clustering on a Single-read Illumina Flow cell. Reads of 50 bases were generated using the TruSeq SBS chemistry on an Illumina HiSeq 4000 or an Illumina NovaSeq 6000 sequencer.