GSE34995 Processing Pipeline

Publication

Cell reports (2012) — PMID 22574288

Dataset

Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins (RNA-Seq)

1. 1

   Strand-specific RNA-seq reads from control siRNA treatment, and each hnRNP depletion experiment were processed as previously described (Polymenidou et al., 2011).

   RNA-seq v2.7.10a (Inferred with models/gemini-2.5-flash)

   $ Bash example

   # Install STAR (if not already installed)
   # conda install -c bioconda star
   
   # Define variables
   # Placeholder for mouse GRCm39 genome index, as the original paper used mouse mm9 and the latest assembly is preferred.
   GENOME_DIR="/path/to/STAR_genome_index/GRCm39"
   READ1="control_siRNA_R1.fastq.gz" # Example input for control siRNA treatment
   READ2="control_siRNA_R2.fastq.gz" # Example input for control siRNA treatment (paired-end assumed)
   OUTPUT_PREFIX="control_siRNA_aligned"
   THREADS=8 # Number of CPU threads to use
   
   # Create genome index (run once for a given genome, uncomment and adjust paths if needed)
   # STAR --runMode genomeGenerate \
   #      --genomeDir ${GENOME_DIR} \
   #      --genomeFastaFiles /path/to/GRCm39.primary_assembly.fasta \
   #      --sjdbGTFfile /path/to/gencode.vM25.annotation.gtf \
   #      --runThreadN ${THREADS}
   
   # Align strand-specific RNA-seq reads using STAR
   # Assuming dUTP-based library prep, which is typically reverse-stranded, 
   # indicated by --outSAMstrandField reverse for the XS tag.
   STAR --genomeDir ${GENOME_DIR} \
        --readFilesIn ${READ1} ${READ2} \
        --runThreadN ${THREADS} \
        --outFileNamePrefix ${OUTPUT_PREFIX}_ \
        --outSAMtype BAM SortedByCoordinate \
        --outSAMstrandField reverse \
        --outFilterMultimapNmax 20 \
        --outFilterMismatchNmax 999 \
        --outFilterMismatchNoverLmax 0.1 \
        --alignIntronMin 20 \
        --alignIntronMax 1000000 \
        --alignMatesGapMax 1000000 \
        --readFilesCommand zcat

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2. 2

   An average of 70% of reads mapped uniquely to our gene structure database, using Bowtie (version 0.12.2, with parameters –l 20 –m 5 –k 5 ––best ––un –– max –q).

   Bowtie v0.12.2 GitHub

   $ Bash example

   # Install Bowtie 0.12.2
   # conda install -c bioconda bowtie=0.12.2
   
   # Note: 'gene_structure_index' refers to a pre-built Bowtie index for the gene structure database.
   # 'input_reads.fastq' is the input FASTQ file containing the reads.
   # 'aligned_reads.sam' will contain the aligned reads in SAM format.
   # 'unaligned_reads.fastq' will contain reads that did not align.
   # The parameter '--max' from the description is not a valid Bowtie 0.12.2 flag and has been omitted.
   
   bowtie -q \
          -l 20 \
          -m 5 \
          -k 5 \
          --best \
          --un unaligned_reads.fastq \
          gene_structure_index \
          input_reads.fastq \
          -S aligned_reads.sam

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Tools Used