GSM8992160: Stat1-ChIP, untreated WT748A, rep.3; Mus musculus; ChIP-Seq
Cross-linking was quenched with 1.375 M glycine for 5 minutes at room temperature. Nuclei were isolated, and chromatin was enzymatically sheared using micrococcal nuclease for 17 minutes at 37 °C. Five percent of the sheared chromatin was reserved as input, and the remainder was subjected to immunoprecipitation using anti–STAT1 antibody (1:50 dilution). Immunocomplexes were reverse cross-linked overnight at 65 °C with Proteinase K, and DNA was purified using phenol/chloroform extraction. ChIP DNA libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer's instructions. Paired-end sequencing (2 × 101 bp) was performed on an Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA). Adapter trimming was conducted with Cutadapt (v4.8), using the following adapter sequences: Read 1: 5′-AGATCGGAAGAGCACACGTCTGAACTCCAGTCA-3′; Read 2: 5′-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3′. Read quality was assessed using FastQC (default settings). Trimmed reads were aligned to the mouse reference genome (mm10) using Bowtie2 (v2.5.3) with the “very sensitive end-to-end” preset. BAM files were further processed using the Galaxy platform. Low-quality reads (MAPQ < 30), improperly paired reads, mitochondrial reads, and PCR duplicates were removed using 'Filter' (v2.4.2) and 'MarkDuplicates' (v3.1.1.0). Peak calling was performed using MACS2 callpeak (v2.2.9.1).