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Sample GSM5709262 Query DataSets for GSM5709262
Status Public on Jul 30, 2023
Title SARSCoV2_3P_Inf_GEX
Sample type SRA
 
Source name Vero E6 African Green Monkey kidney cell line
Organism Chlorocebus aethiops
Characteristics cell line: Vero E6 African Green Monkey kidney cell line
infection: SARS-CoV-2, MOI 0.1, 24 hours
scrnaseq method: 10X Genomics Chromium Next GEM Single Cell 3' v3.1
sequencing method: Read 1 28 nt, i7 index 8 nt, read 2 130 nt
Treatment protocol In 6 well plates, cells were infected with SARS-CoV-2 (USA-WA1/2020) at an MOI of 0.1 or mock infected with equivalent control media in reduced serum media (2% FBS, 1% PSN) for 24 hours.
Growth protocol Vero E6 cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PSN) at 37°C and 5% CO2.
Extracted molecule total RNA
Extraction protocol Cultures were washed with calcium/magnesium-PBS and disassociated with TrypLE (Gibco # 12605010) for 5 minutes at 37°C. Cells were then centrifuged (200 x g for 5 minutes), resuspended in calcium/magnesium free PBS supplemented with 1% BSA and counted. Cells were then filtered through a 40μm FlowMi strainer (ScienceWare # H13680-0040) and counted again. Mock and infected samples were loaded on separate lanes of a 10X Genomics Chromium Controller, according to manufacturer's protocol, for either NextGem Single Cell 3' v3.1 (10X 3') or NextGEM Single Cell V(D)J v1.1 (10X 5').
Single cell RNA sequencing of mock and SARS-CoV-2 infected Vero E6 cells performed with either 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') or 10X Genomics Chromium Next GEM Single Cell V(D)J (10X 5'). Final 10X 3' mock and infected gene expression libraries and 10X 5' infected gene expression libraries were PCR amplified for 16 cycles, while the 10X 5' mock gene expression library was amplified for 14 cycles. 10X 3' libraries were pooled and sequenced according with the following read lengths: read1 28 nt, i7 index 8 nt, read2 130 nt. 10X 5' libraries were also pooled and sequenced with the following read lengths: read 1 26 nt, i7 index 8nt, read 2 132 nt. 10X 5' were also sequenced with our custom extended R1 protocol (read 1 158 nt, i7 index 8 nt, no read 2).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Data processing Fastq files from bcl files for standard 10X 3' and 10X 5' libraries were generated using cellranger (v3.1.0, 10X Genomics) mkfastq. Fastqs for 5' libraries sequenced with the Extended R1 protocol were generated using bcl2fastq (v2.20.0, Illumina, Inc).Extended R1 fastqs were then separated into pseudo R1 fastqs, containing the cell barcode and UMI, and pseudo read 2 (R2) fastqs, containing cDNA sequence, using a customized Python/3.7.3 script (available at github link pending) as follows. The cell barcode and UMI are selected from the first 26 bp of R1. The subsequent 13 bp derive from the template switch oligonucleotide and are ignored. The remaining nucleotides (and corresponding quality scores) are reverse complemented and stored as pseudo R2. The read header of the pseudo R2 fastqs are modified to reflect the format for standard R2 fastqs.
To control for differences in sequencing depth per library, read depth per library was downsampled to approximately 50,000 reads multiplied by the number of cells in the library using seqtk (version 1.2). We do this by generating a whitelist of cell barcodes using a preliminary gene cell matrix generated using cellranger (v3.1.0, 10X Genomics) count with a combined African Green Monkey reference (ChlSab1.1) combined with SARS-CoV-2 transcripts (NC_045512.2) with modifications for the USA/WA01 strain. This output was analyzed in R (v4.0.4) with the Seurat package (version 4.0.1) to filter out putative multiplets, empty droplets, and dead cells based on total UMIs/cell, number of genes/cell, and percent of mitochondrial gene expression per cell.
Downsampled fastq files were mapped and quantified using cellranger (v3.1.0, 10X Genomics) count to a combined African Green Monkey (ChlSab1.1) and SARS-CoV-2 reference derived from empirically defined RNAs sequenced with long-read direct RNA Nanopore sequencing published by Kim et al, Cell 2020. These were downloaded from the UCSC Genome Table Browser after filtering for TRS-dependent transcripts and score > 900 and exporting to gtf format. Transcripts for previously unknown ORFs were excluded and an additional annotation for genomic RNA covering the entire length of the SARS-CoV-2 genome was added. Modifications the reference were made to match the USA/WA01 SARS-CoV-2 strain.
To quantify SARS-CoV-2 RNAs, we developed single cell coronavirus sequencing (scCoVseq), which quantifies only unambiguous genomic RNA (gRNA)-derived reads and reads from subgenomic mRNAs (sgmRNAs) containing junctions between the 5' leader of the genome and sgmRNA. We did this using samtools (v1.11) to filter SARS-CoV-2 aligned reads to contiguous reads aligning upstream of sgmRNA-containing regions of the genome or to SARS-CoV-2 reads aligning in part the the 5' leader (defined as the 5' proximal 80 nt of the genome) and in part to the regions of the genome used by sgmRNAs with any gap length. Reads not matching these filters were excluded. Filtered SARS-CoV-2 reads were then merged with host reads and quantified using umi_tools (v1.0.0) and converted to a sparse matrix using a custom R (v3.5.3) script with the Matrix package (v1.2-18) and the readr package (v1.3.1). UMIs assigned to multiple genes were removed from the resulting matrix during downstream analysis.
Genome_build: custom build described above
Supplementary_files_format_and_content: h5: gene x cell matrix output by cellranger in h5 format; rds: gene x cell matrix output by scCoVseq as R data file, rds;
 
Submission date Nov 30, 2021
Last update date Jul 30, 2023
Contact name Brad R Rosenberg
E-mail(s) brad.rosenberg@mssm.edu
Organization name Icahn School of Medicine at Mount Sinai
Department Department of Microbiology
Street address 1 Gustave L Levy Place, Box 1124
City New York
State/province NY
ZIP/Postal code 10029
Country USA
 
Platform ID GPL22096
Series (1)
GSE189900 Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single cell RNA sequencing
Relations
BioSample SAMN23522407
SRA SRX13263698

Supplementary file Size Download File type/resource
GSM5709262_SARSCoV2_3P_Inf_GEX_CellRanger_Output_raw_feature_bc_matrix.h5 119.3 Mb (ftp)(http) H5
GSM5709262_SARSCoV2_3P_Inf_GEX_scCoVseq_Output_ThreeP_Inf_Kim_Single_Chrom_Gene_Cell_Matrix.rds.gz 2.1 Mb (ftp)(http) RDS
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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