ChIP-seq of HA-tagged klf7 in N2A cells (klf7-rep2)

Klf7 was cloned into pIRM-3*HA vector according to the manufacturer's instructions and transfected into N2A cells. 48 hours later, N2A cells were harvested. The cells were crosslinked with 1% formaldehyde for 10 minutes and neutralized with glycine (150 mM final). The cells were lysed, chromatin was sheared into ~300 bp fragments by ultrasonic treatment. Immunoprecipitation was performed using a ChIP-grade anti-HA antibody (Abcam, ab9110) and isotype control antibody (Abcam, ab172730) overnight at 4°C, and the samples were incubated with magnetic beads. The bound proteins were eluted from the beads, and reverse crosslinking was carried out overnight at 65°C. Phenol-chloroform was used to extract DNA for sequencing.  

ChIP DNA degradation and contamination was monitored on agarose gels. DNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). DNA concentration was measured using Qubit® DNA Assay Kit in Qubit® 3.0 Flurometer (Life Technologies, CA, USA). The purified DNA was used for ChIP-seq library preparation. Subsequently, pair-end sequencing of sample was performed on Illumina platform (Illumina, CA, USA). Library quality was assessed on the Agilent Bioanalyzer 2100 system.

Raw data (raw reads) of fastq format were firstly processed using fastp (version 0.19.11) software. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Clean reads were aligned to the mouse reference genome (Ensemble_GRCm38.94) for each ChIP-seq experiment in N2A cells using BWA mem (v 0.7.12). After mapping reads to the reference genome, we used the MACS2 (version 2.1.0) peak calling software to identify regions of IP enrichment over background. A q-value threshold of 0.05 was used for all data sets. After peak calling, the distribution of chromosome distribution, peak width, fold enrichment, significant level and peak summit number per peak were all displayed.  Homer was used to detect the denovo sequence motif and the matched known motifs. Reproducibility was defined as overlapping peaks between two biological ChIP-seq replicates.