GSE130238 Processing Pipeline

Publication

Cell stem cell (2019) — PMID 31474560

Dataset

Nested oscillatory dynamics in cortical organoids model early human brain network development

1. 1

   base-calling: cellranger mkfastq (version 2.1.1)

   Cell Ranger v2.1.1

   $ Bash example

   # Download Cell Ranger 2.1.1 from 10x Genomics website (replace with actual download link if available for this specific version)
   # wget https://cf.10xgenomics.com/releases/cell-ranger/cellranger-2.1.1.tar.gz
   # tar -xzf cellranger-2.1.1.tar.gz
   # export PATH=/path/to/cellranger-2.1.1:$PATH
   
   # Define input and output paths
   BCL_RUN_FOLDER="/path/to/illumina/run/folder" # e.g., /sequencer/data/171101_SN001_0001_AABCD_run
   OUTPUT_ID="my_mkfastq_output"
   SAMPLE_SHEET_CSV="/path/to/sample_sheet.csv" # A CSV file describing samples and their barcodes
   
   # Run cellranger mkfastq to demultiplex BCL files into FASTQ files
   cellranger mkfastq --run "${BCL_RUN_FOLDER}" \
                      --id "${OUTPUT_ID}" \
                      --csv "${SAMPLE_SHEET_CSV}"

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2. 2

   alignment, cell barcode processing, umi processing, feature-barcode matrix generation: cellranger count (version 2.1.1)

   Cell Ranger v2.1.1

   $ Bash example

   # Cell Ranger version 2.1.1 installation (example)
   # Download the Cell Ranger 2.1.1 tarball from 10x Genomics website (requires login or specific release URL)
   # wget https://cf.10xgenomics.com/releases/cell-exp/cellranger-2.1.1.tar.gz
   # tar -xzf cellranger-2.1.1.tar.gz
   # export PATH=/path/to/cellranger-2.1.1:$PATH
   
   # Prepare Cell Ranger compatible transcriptome reference (example: Human GRCh38)
   # Download from 10x Genomics website:
   # wget https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-GRCh38-2020-A.tar.gz
   # tar -xzf refdata-gex-GRCh38-2020-A.tar.gz
   REF_TRANSCRIPTOME="/path/to/refdata-gex-GRCh38-2020-A" # Replace with actual path to your transcriptome reference
   
   # Specify the directory containing FASTQ files
   FASTQ_DIR="/path/to/your/fastq_directory" # Replace with actual path to your FASTQ files
   
   # Define a unique ID for this run
   RUN_ID="my_10x_experiment_id" # Replace with a unique identifier for this analysis
   
   # Specify the sample name(s) as they appear in the FASTQ file names (e.g., 'sample1_S1_L001_R1_001.fastq.gz' -> 'sample1')
   SAMPLE_NAME="your_sample_name" # Replace with the actual sample name(s) if different from the directory structure
   
   # Execute cellranger count
   cellranger count \
       --id="${RUN_ID}" \
       --transcriptome="${REF_TRANSCRIPTOME}" \
       --fastqs="${FASTQ_DIR}" \
       --sample="${SAMPLE_NAME}" \
       --expect-cells=3000 \
       --localcores=8 \
       --localmem=64

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3. 3

   Supplementary files including lists of barcodes, genes, and feature-barcode matrix were generated by Cell Ranger v2.1.1.

   Cell Ranger v2.1.1

   $ Bash example

   # Install Cell Ranger (example, adjust for specific system/version)
   # wget https://cf.10xgenomics.com/releases/cell-exp/cellranger-2.1.1.tar.gz
   # tar -xzf cellranger-2.1.1.tar.gz
   # export PATH=/path/to/cellranger-2.1.1:$PATH
   
   # Define variables for the run
   RUN_ID="my_cellranger_run"
   FASTQ_DIR="/path/to/your/fastq_files"
   TRANSCRIPTOME_REF="/path/to/10x_genomics_refdata/refdata-gex-GRCh38-2020-A" # Placeholder for a common human transcriptome
   SAMPLE_NAME="my_sample"
   EXPECTED_CELLS="3000" # Placeholder, adjust based on experiment
   
   # Run Cell Ranger count to generate feature-barcode matrix, barcode lists, and gene lists
   cellranger count \
       --id="${RUN_ID}" \
       --transcriptome="${TRANSCRIPTOME_REF}" \
       --fastqs="${FASTQ_DIR}" \
       --sample="${SAMPLE_NAME}" \
       --expect-cells="${EXPECTED_CELLS}"

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