title: GSM8171564: SH-SY5Y, WT, rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: 1 × 107 cells in culture dishes were fixed with 1 % formaldehyde at room temperature for 10 minutes, and the fixation was stopped via addition of 0.125 M glycine at room temperature for 5 minutes. The cells were washed with cold PBS and scraped into cold SDS lysis buffer from a ChIP Assay Kit (P2078, Beyotime, China). Then, the Covaris™ S220 (Covaris, Amercia) was used to sonicate the sample for a total of 8 minutes (with intervals) at 4 ℃.The chromatin pellets were extracted from cells by lysis buffer from a ChIP Assay Kit (P2078, Beyotime, China). Sonicated chromatin was then incubated with 2 μg of anti-histone H4K5la (PTM-1407RM, PTM BIO, China) or control IgG (66362S, CST, Amercia) precoated on Dynabeads protein G (10007D, Dynabeads protein G, Amercia) at 4 ℃ overnight. The spike-in antibody (61686, active motif, Amercia) and spike-in chromatin (53083, active motif, Amercia) were added as internal control. Immunoprecipitants were washed sequentially with low salt immune complex wash buffer, high salt immune complex wash buffer, LiCl immune complex wash buffer and TE buffer (P2078, Beyotime, China). Chromatin precipitated was then eluted by protease K digestion, cross-link reversed at 65 ℃ for 4 hours, and purified using a QIAquick PCR purification kit (51304, Qiagen, Germany). 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. 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. The single A overhang enables ligation to adaptors with single T overhangs. Adaptor containing sequences required downstream in the sequencing workflow are ligated to the dA-tailed fragment.  The KAPA HiFi DNA Polymerase has been inactivated, enabling amplification of DNA.