GSM5625173: nucleus pulposus cells_input; Homo sapiens; ChIP-Seq
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. ChIP-Sequencing library preparation was performed according to Illumina's protocol Preparing Samples for ChIP Sequencing of DNA.    Library Preparation: 10 ng DNA of each sample was converted to phosphorylated blunt-ended; An 'A' base was added to the 3' end of the blunt phosphorylated DNA fragments; Illumina's genomic adapters were ligated to the A tailed DNA fragments; PCR amplification was performed to enrich ligated fragments. The enriched product of ~200-1500 bp was size selected using AMPure XP beads.    Sequencing: The library was denatured with 0.1M NaOH to generate single-stranded DNA molecules, and loaded onto channels of the flow cell at 8pM concentration, amplified in situ using Cluster Kit. Sequencing was carried out by running 2×150 cycles on Illumina NovaSeq 6000 according to the manufacturer's instructions.    Data Analysis: After the sequencing platform generated the sequencing images, the stages of image analysis and base calling were performed using Off-Line Basecaller software (OLB V1.8). Sequence quality was examined using the FastQC software. After passing Solexa CHASTITY quality filter, the clean reads were aligned to human reference genome UCSC HG19 using BOWTIE (V2.2.7). Aligned reads were used for peak calling of the ChIP regions using MACS V1.4.2. Statistically significant ChIP-enriched regions (peaks) were identified by comparison of IP vs Input or comparison to a Poisson background model, using a p-value threshold of 10-4. Then the peaks were annotated by the nearest gene using the newest UCSC RefSeq database.