title: GSM8377447: SK-N-SH, DA+; Homo sapiens; ChIP-Seq
library_construction_protocol: 1 x 107 SK-N-SH cells in culture dishes were fixed with 1% formaldehyde at room temperature for 10 min, and the fixation was stopped via addition of cool 0.125 M glycine solution at room temperature for 5 min. The cells were collected and washed with PBS and scraped into cold lysis buffer (10 mM tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% NP-40). Chromatin pellets obtained by centrifugation were resuspended with 0.8 ml of RIPA buffer (50 mM tris-HCl, pH 7.5, 300 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS, and 1 × protease inhibitor cocktail) and sonicated at 25% amplitude for a total of 8 min (with intervals). Sonicated chromatin was then incubated with 2 mg of anti-histone H4Q27dop or control IgG precoated on protein A/G magnetic beads (Thermo fisher, no. 88803) at 4°C overnight. Immunoprecipitants were washed twice with RIPA buffer and additional three times with LiCl washing buffer (100 mM tris-HCl, pH 7.5, 500 mM LiCl, 1% NP-40, and 1% sodium deoxycholate). Chromatin precipitated was then eluted by protease K digestion, cross-link reversed at 65°C overnight, and purified using a QIAquick PCR purification kit (Qiagen). The immunoprecipitated DNA fragments were either sent for high-throughput sequencing (Novogene) or assessed by qPCR as described above. To normalize the sequencing data, samples were standardized by the inclusion of Drosophila DNA as a spike-in control 1.DNA fragment Adjust the volume of the combined gDNA sample to a total volume of 41 μL using 1×TE and fragment the DNA to 300 bp using the Biorupter.  2.End repair Blunt-ended fragments are created by filling in or chewing back 3' and 5' overhangs. Phosphorylation of the 5' ends ensures the fragments are suitable for ligation. 3.dA-Tailing This step is dA-tailing of the 3'end of the end-repaired, phosphorylated fragments. This single A overhang enables ligation to adaptors with single T overhangs. 4.Adaptor ligation Adaptor containing sequences required downstream in the sequencing workflow are ligated to the dA-tailed fragment.  5.PCR amplification The KAPA HiFi DNA Polymerase has been inactivated, enabling amplification of DNA. 6.2 % agarose gel select the fragment 200~500bp.