|Public on Aug 08, 2019
|sgRNA perturb-seq experiment (gemgroup 1)
|cell type: K562 chronic myelogenous leukemia cell line
|K562 cells expressing dCas9-KRAB were transduced with lentivirus carrying the sgRNA expression cassettes and a BFP marker. Transduced cells were isolated by FACS 3 days after transduction and grown for 2 additional days. Growth was performed in RPMI media supplemented with FBS and Pen/Strep/Gln.
|Single cells were lysed in droplets using the 10X Chromium instrument
Reverse transcription was performed in droplet and library construction was performed according to the 10X Chromium Single Cell 3' Reagent Kit User Guide (v2 chemistry)
|Illumina NovaSeq 6000
|Single-cell RNA-seq data, gemgroup 1
Single-cell RNA-seq data, gemgroups 1-8 aggregated and collapsed to UMI tables:
|Fastq.gz files were generated from raw Illumina BCL files using bcl2fastq
Reads were aligned and corrected from each fastq.gz files using 10X Genomics Cellranger version 2.1.1 with default paramaters (cellranger count), resulting in the "molecule_info.h5" HDF5 files
Reads from multiple channels (gemgroups) of the 10X run were combined using 10X Genomics Cellranger version 2.1.1 (cellranger aggr)
Gene-barcode matrices were determined using 10X Genomics Cellranger version 2.1.1 with default parameters, producing the matrix market exchange files (the UMI matrices) and the TSV files (which are the row and column names of the UMI matrix)
Guide barcode identities were determined as described in the paper from targeted amplicon sequencing, resulting in the "cell_identities.csv" files. Identities were called for all cell barcodes called by cellranger in the previous step.
Supplementary_files_format_and_content: The experiment consists of one large pooled experiment of ~100,000 single cells that was processed as 8 independent replicate samples (corresponding to lanes on the 10X Chromium machine, and encoded here by gemgroup.) All sequencing data ultimately derive from the same experiment, so they are merged during the creation of the processed data files. Gene-barcode matrices are the UMI-collapsed counts of these reads, representing counts of molecules per cell as determined and filtered by cellranger. These are provided in the format outputted by cellranger: the matrix itself is in matrix market exchange format, with the rows (cell barcodes) and columns (gene identities) labels given in TSV format as described in the cellranger documentation. We have uploaded both the "filtered" and "raw" versions of these files. Guide barcodes were specifically amplified from the RNA-seq libraries and sequenced to higher coverage, and then identities were called as discussed in the paper. These are simply formatted as tables that identify a guide barcode for each of the sets of files from the previous step.
|Jun 26, 2019
|Last update date
|Aug 09, 2019
|University of California, San Francisco
|Biochemistry Box 2542, 1700 4th St QB3 Rm 404
|Exploring genetic interaction manifolds constructed from rich phenotypes