GSE217735 Processing Pipeline

Publication

Neuron (2024) — PMID 38697111

Dataset

Epigenetics CRISPRi screen in mouse neuronal progenitor cells (NPCs)

1. 1

   Illumina RTA was used to create .bcl files, and bcl2fastq v2.20.0.422 software was used for base calling (for samples split between flowcells and later merged per run)

   bcl2fastq v2.20.0.422 GitHub

   $ Bash example

   # Install bcl2fastq (typically downloaded from Illumina's website or via conda)
   # conda create -n bcl2fastq_env bcl2fastq=2.20.0.422
   # conda activate bcl2fastq_env
   
   # Define the Illumina run folder (top-level directory containing BCL files, SampleSheet.csv, etc.)
   # Replace with the actual path to your Illumina run folder
   ILLUMINA_RUN_FOLDER="/path/to/illumina/run/folder"
   
   # Define the output directory for demultiplexed FASTQ files
   # Replace with your desired output path
   OUTPUT_FASTQ_DIR="/path/to/output/fastqs"
   
   # Create the output directory if it doesn't exist
   mkdir -p "${OUTPUT_FASTQ_DIR}"
   
   # Execute bcl2fastq for base calling and demultiplexing
   # bcl2fastq will read the SampleSheet.csv from the run folder by default
   bcl2fastq --runfolder-dir "${ILLUMINA_RUN_FOLDER}" \
             --output-dir "${OUTPUT_FASTQ_DIR}" \
             --no-lane-splitting # Use this if you want to merge data from different lanes into a single sample file
   

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2. 2

   Raw reads were mapped against sgRNA library and raw counts table of sgRNA was generated using MAGeCK v0.5.9.3

   MAGeCK v0.5.9.3

   $ Bash example

   # Install MAGeCK (if not already installed)
   # conda install -c bioconda mageck
   
   # Assuming 'sgRNA_library.txt' is the sgRNA library definition file
   # and 'sample1.bam', 'sample2.bam' are the alignment files generated from mapping raw reads.
   # The actual sample IDs and BAM files would need to be specified based on the experiment.
   
   mageck count \
     -l sgRNA_library.txt \
     --sample-id sample1,sample2 \
     --bam-file sample1.bam,sample2.bam \
     -n sgRNA_counts

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