GSE253825 Processing Pipeline

Publication

Neuron (2024) — PMID 38697111

Dataset

Nonsense-mediated mRNA decay counteracts TRP53 to maintain the progenitor cell pool for brain development (snRNA-seq_e15invivo)

1. 1

   STAR (version 2.7.11a, reference genome: mm10-2020-A<based on Gencode vM23, and filtering out pseudogenes, TEC, miRNA etc. a total of 32285 genes) was used to perform alignment, filtering, barcode counting, and UMI counting for single cell RNA-Seq.

   STAR v2.7.11a

   $ Bash example

   # Install STAR (if not already installed)
   # conda install -c bioconda star
   
   # Define variables
   # Path to the custom STAR index built with mm10, Gencode vM23, and filtered genes
   GENOME_DIR="path/to/STAR_index_mm10_Gencode_vM23_filtered"
   # Input FASTQ files (e.g., R1 containing UMI/barcode, R2 containing cDNA)
   READ1_FASTQ="path/to/sample_R1.fastq.gz"
   READ2_FASTQ="path/to/sample_R2.fastq.gz"
   # Output directory prefix
   OUTPUT_PREFIX="star_alignment_output/"
   # Path to the cell barcode whitelist (specific to scRNA-seq chemistry, e.g., 10x Genomics)
   CB_WHITELIST="path/to/cell_barcode_whitelist.txt"
   
   # Create output directory
   mkdir -p "${OUTPUT_PREFIX}"
   
   # Run STAR for alignment, filtering, barcode counting, and UMI counting
   # Note: --solo parameters (soloType, soloCBwhitelist, soloCBstart, soloCBlen, soloUMIstart, soloUMIlen)
   #       must be adjusted based on the specific single-cell RNA-Seq library preparation chemistry.
   #       The example below assumes a common 10x Genomics v3 setup (16bp CB, 12bp UMI in R1).
   STAR --genomeDir "${GENOME_DIR}" \
        --readFilesIn "${READ1_FASTQ}" "${READ2_FASTQ}" \
        --readFilesCommand zcat \
        --outFileNamePrefix "${OUTPUT_PREFIX}" \
        --outSAMtype BAM SortedByCoordinate \
        --outSAMunmapped Within \
        --outFilterMultimapNmax 1 \
        --outFilterScoreMinOverLread 0.3 \
        --outFilterMatchNminOverLread 0.3 \
        --outFilterMismatchNmax 3 \
        --limitBAMsortRAM 60000000000 \
        --soloType CB_UMI_Simple \
        --soloCBwhitelist "${CB_WHITELIST}" \
        --soloCBstart 1 --soloCBlen 16 \
        --soloUMIstart 17 --soloUMIlen 12 \
        --soloFeatures Gene Velocyto \
        --soloCellFilter CellRanks 30 0.99 10000 \
        --soloOutFileNames Features.mtx Features.barcodes.txt Features.genes.txt

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Tools Used