GSE273094 Processing Pipeline

Publication

Proceedings of the National Academy of Sciences of the United States of America (2024) — PMID 39141348

Dataset

Zfp697 is an RNA-binding protein that regulates skeletal muscle inflammation and remodeling (Zfp697 transduced primary mouse myotubes ChIP-Seq)

1. 1

   Reads were aligned to the GRCm38.p6 version of the mouse genome using STAR and duplicated reads were removed with MarkDuplicates.

   STAR v2.7.10a GitHub

   $ Bash example

   # Install STAR (example)
   # conda install -c bioconda star
   
   # Install Picard (example)
   # conda install -c bioconda picard
   
   # Define variables
   # Replace with actual paths to your reference genome and input reads
   GENOME_DIR="path/to/STAR_index/GRCm38.p6" # Directory containing STAR genome index
   GENOME_FASTA="path/to/reference/GRCm38.p6.fa" # Reference FASTA for index generation
   GENOME_GTF="path/to/annotation/GRCm38.p6.gtf" # GTF for splice junction annotation (recommended for RNA-seq)
   READS_R1="sample_R1.fastq.gz" # Input R1 FASTQ file
   READS_R2="sample_R2.fastq.gz" # Input R2 FASTQ file (remove if single-end)
   OUTPUT_PREFIX="sample_aligned" # Prefix for output files
   ALIGNED_BAM="${OUTPUT_PREFIX}Aligned.sortedByCoordinate.out.bam"
   DEDUP_BAM="${OUTPUT_PREFIX}.deduplicated.bam"
   DEDUP_METRICS="${OUTPUT_PREFIX}.deduplication_metrics.txt"
   NUM_THREADS=8 # Number of threads to use for STAR
   PICARD_JAR="picard.jar" # Path to picard.jar, or just "picard.jar" if in PATH
   
   # --- STAR Alignment ---
   # Generate STAR genome index (run once per genome version)
   # STAR --runMode genomeGenerate \
   #      --genomeDir "${GENOME_DIR}" \
   #      --genomeFastaFiles "${GENOME_FASTA}" \
   #      --sjdbGTFfile "${GENOME_GTF}" \
   #      --runThreadN "${NUM_THREADS}"
   
   # Align reads to the GRCm38.p6 mouse genome
   STAR --genomeDir "${GENOME_DIR}" \
        --readFilesIn "${READS_R1}" "${READS_R2}" \
        --runThreadN "${NUM_THREADS}" \
        --outFileNamePrefix "${OUTPUT_PREFIX}" \
        --outSAMtype BAM SortedByCoordinate \
        --outSAMunmapped Within \
        --outSAMattributes All \
        --outFilterMultimapNmax 20 \
        --outFilterMismatchNmax 999 \
        --outFilterMismatchNoverLmax 0.1 \
        --alignIntronMin 20 \
        --alignIntronMax 1000000 \
        --alignMatesGapMax 1000000 \
        --limitBAMsortRAM 30000000000 # Adjust based on available RAM (e.g., 30GB)
   
   # --- MarkDuplicates (Picard) ---
   # Remove duplicated reads
   java -jar "${PICARD_JAR}" MarkDuplicates \
        I="${ALIGNED_BAM}" \
        O="${DEDUP_BAM}" \
        M="${DEDUP_METRICS}" \
        REMOVE_DUPLICATES=true \
        ASSUME_SORTED=true

   Copy View on GitHub
2. 2

   We used MACS3 callpeak for peak calling (q- value < 0.05 and fold change ≥ 1.5).

   MACS2

   $ Bash example

   # Install MACS3 (example using conda)
   # conda install -c bioconda macs3
   
   # Define input and output files
   TREATMENT_BAM="treatment.bam" # Replace with your actual treatment/IP BAM file
   CONTROL_BAM="control.bam"   # Replace with your actual control/input BAM file
   OUTPUT_PREFIX="my_peaks"    # Prefix for output files
   OUTPUT_DIR="./"
   
   # Run MACS3 callpeak
   macs3 callpeak \
     -t "${TREATMENT_BAM}" \
     -c "${CONTROL_BAM}" \
     -f BAM \
     -g hs \
     -n "${OUTPUT_PREFIX}" \
     --outdir "${OUTPUT_DIR}" \
     -q 0.05 \
     --fold-enrichment 1.5

   Copy
3. 3

   Bedtools were used to generate coverage tracks.

   bedtools v2.31.0 (Inferred with models/gemini-2.5-flash) GitHub

   $ Bash example

   # Install bedtools if not already installed
   # conda install -c bioconda bedtools
   
   # Example: Generate a bedGraph file representing genome-wide coverage from a BAM file.
   # This bedGraph can then be converted to a bigWig file for visualization as a coverage track.
   # Input:
   #   - aligned_reads.bam: A sorted and indexed BAM file containing aligned sequencing reads.
   # Output:
   #   - coverage.bedgraph: A bedGraph file showing per-base coverage depth across the genome.
   
   bedtools genomecov -ibam aligned_reads.bam -bg > coverage.bedgraph

   Copy View on GitHub

Tools Used