title: GSM5957385: ChIP IP H3K27ac Interzone Rep2; Gallus gallus; ChIP-Seq
library_construction_protocol: For the preparation of ChIPseq libraries, the histone-DNA complexes were immunoprecipitated with antibody. For each sample, 100 specimens were collected from the 3rd hindlimb digit of developing chicken ebmryo (HH32) and pooled together. Further, samples were dissociated using Collagenase-II and passed through a 40-m cell strainer. The 1mln of cells were cross-linked and after series of washes the cell nuclei were further isolated. Subsequently, the nuclei were treated with SDS-containing lysis buffer, and chromatin was sheard obtaining the average size of 150 bp. For ChIP, 500 ng of sonicated chromatin was immunoprecipitated with 7.5 g of antibody. Input sample was collected prior to immunoprecipitation reaction. ChIP-seq libraries were prepared using QIAseq Ultra Low Input Library Kit (QIAGEN, Hilden, Germany). Briefly, DNA was end-repaired, adenosines were added to the 3 ends of dsDNA and adapters were ligated. Adapters containing DNA fragments were amplified by PCR using NEB starters. Mean library size was 300 bp. Libraries were run in the rapid run flow cell and were single-end sequenced (65 bp) on HiSeq 1500 (Illumina, San Diego, CA 92122 USA).