GSM8884970: CHI_INPUT; Homo sapiens; ChIP-Seq
RNAseq: After treatment, the samples were extracted using Trizol. The purity and concentration of the samples were assessed with a NanoDrop 2000 spectrophotometer, while the integrity of RNA samples was evaluated using the LabChip GX Touch microfluidic capillary system. ChIPseq: The cells were fixed with 1% formaldehyde at room temperature for 10 minutes. Fixation was subsequently quenched by the addition of 2 mL of 10× glycine, followed by an additional 5 minutes of incubation at room temperature. The cells were then scraped, lysed, digested, and sheared via sonication. Lysates were clarified by centrifugation, and 2% of the supernatant was reserved as the input sample, which could be stored at –20 °C for future use. The supernatant was incubated overnight at +4 °C with immunoprecipitating antibodies, specifically antibodies against H3K27me3 and H3. Following this incubation, 30 μL of Protein G Magnetic Beads were added to each immunoprecipitation reaction, which was then rotated and incubated for 2 hours at 4 °C. After washing the beads, chromatin was eluted from the antibody/protein G magnetic beads. For all samples, including the 2% input sample obtained from previous steps, crosslinks were reversed by the addition of 6 μL of 5 M NaCl and 2 μL of Proteinase K, followed by a 2-hour incubation at 65 °C. DNA purification was performed using spin columns. The purity of the DNA was assessed using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), while the DNA concentration was quantified using the Qubit® DNA Assay Kit in a Qubit® 3.0 Fluorometer (Life Technologies, CA, USA). The purified DNA was subsequently employed for ChIP-seq library preparation. RNAseq: After passing the quality assessment, total RNA samples were enriched for eukaryotic mRNA using Oligo(dT) magnetic beads, followed by mRNA fragmentation using Fragmentation Buffer. First- and second-strand cDNA were synthesized using mRNA as a template, and the resulting cDNA was purified. The purified double-stranded cDNA underwent end repair, A-tailing, and adapter ligation, and fragments of approximately 350 bp were selected and recovered. Finally, PCR amplification was performed to obtain the cDNA library. ChIPseq :Quality Control Raw data (raw reads) in FASTQ format were first processed using fastp (version 0.19.11, Chen et al., 2018) software. In this step, clean data (clean reads) were obtained by removing reads containing adapters, reads with poly-N, and low-quality reads from the raw data. Additionally, the Q20, Q30, and GC content of the clean data were calculated. All downstream analyses were performed based on the clean data with high quality.  Reads Mapping to the Reference Genome Reference genome and gene model annotation files were directly downloaded from the genome website. The index of the reference genome was built using BWA (v0.7.12), and clean reads were aligned to the reference genome using BWA mem (v0.7.12).  Fragment Size Estimation For a specific ChIP-seq binding site, individual reads mapping to the plus or minus strand and showing significant enrichment were considered. For single-end sequencing, the fragment size was estimated using the MACS2 (Zhang et al., 2008) predicted method with default parameters. MACS2 scans the entire genome with a specific window size and calculates the read enrichment level. A specific number of windows were used as samples to build an enrichment model and predict the fragment size. The subsequent peak calling analysis was based on this predicted fragment size.  Peak Detection After mapping reads to the reference genome, MACS2 (version 2.1.0) peak calling software was used to identify regions of immunoprecipitation (IP) enrichment over background. A q-value threshold of 0.05 was applied for all datasets. After peak calling, the distribution of chromosome locations, peak width, fold enrichment, significance level, and the number of peak summits per peak were displayed.