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Sample GSM4546505 Query DataSets for GSM4546505
Status Public on Sep 08, 2020
Title Pat3_cHCV_13
Sample type SRA
 
Source name Cryopreserved PBMCs, Department of Medicine II of the University Hospital Freiburg, Germany
Organism Homo sapiens
Characteristics disease state: chronic HCV infection
tissue: Peripheral Blood
cell type: HCV-specific CD8+ T cells
Extracted molecule polyA RNA
Extraction protocol PBMCs from EDTA anticoagulated patient blood were isolated by density gradient centrifugation using Pancoll (Pan-Biotech, Germany). Magnetic bead-based enrichment of antigen-specific CD8 T cells was further performed. For magnetic bead-based enrichment of HCV-specific CD8+ T cells PBMCs were incubated with peptide-loaded HLA- tetramers coupled to phycoerythrin (PE). Subsequent enrichment was performed with anti-PE beads using the MACS technology (Miltenyi Biotec, Germany) according to the manufacturer’s protocol. Enriched HCV-specific CD8+ T cells were used for multiparametric flow cytometry and transcriptome analysis. Epitope-specific HLA tetramers were generated by conjugation of biotinylated peptide-MHC class I monomers with PE-conjugated streptavidin at a HLA:Strepatividin molar ratio of 5:1. The following peptide-loaded HLA tetramers were used: NS31073(CINGVCWTV)/HLA-A*02:01; NS31406(KLVALGINAV/KLSGLGLNAV)/HLA-A*02:01; NS52594(ALYDVVTKL)/HLA-A*02:01; and NS5B2841(ARMILMTHF)/HLA-B*27:05.
As described in mCEL-Seq2 protocol (Hashimshony et al. 2016 and Herman et al. 2018)
Adapted from TruSeq Small RNA Library Preparation Protocol
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 3000
 
Data processing For image aquisition, intensity extraction and basecalling HiSeq Control Software 2.0.2, RTA 2.4.11 / Recipe Fragment 2.0.0.2​ was used. Conversion of bcl2fastq files was performed using bcl2fastq 2.17.1.14
Paired end reads were aligned to the transcriptome using bwa (version 0.6.2-r126) with default parameters. The transcriptome contained all gene models based on the mouse ENCODE VM9 release downloaded from the UCSC genome browser comprising 57,207 isoforms derived from 57,207 gene loci with 57,114 isoforms mapping to fully annotated chromosomes (1 to 19, X, Y, M). All isoforms of the same gene were merged to a single gene locus. Furthermore, gene loci overlapping by >75% were merged to larger gene groups. This procedure resulted in 34,111 gene groups.
The right mate of each read pair was mapped to the ensemble of all gene loci and to the set of 92 ERCC spike-ins in sense direction. Reads mapping to multiple loci were discarded. The left read contains the barcode information: the first six bases correspond to the cell specific barcode followed by six bases representing the unique molecular identifier (UMI). The remainder of the left read contains a poly(T) stretch. The left read was not used for quantification. For each cell barcode, the number of UMIs per transcript was counted and aggregated across all transcripts derived from the same gene locus.
Based on binomial statistics, the number of observed UMIs was converted into transcript counts (Gruen et al., 2014).
Genome_build: ENCODE VM9
Supplementary_files_format_and_content: CSV files, columns represent each cell barcode (total barcodes used = 192), rows represent the geneid and the values in the file are the quantified number of transcripts.
 
Submission date May 11, 2020
Last update date Sep 09, 2020
Contact name Sagar -
E-mail(s) sagar@uniklinik-freiburg.de
Organization name University Medical Center Freiburg
Department Department of Internal Medicine II
Lab Sagar
Street address Hugstetter Straße 55
City Freiburg
ZIP/Postal code 79106
Country Germany
 
Platform ID GPL21290
Series (1)
GSE150305 Memory-like HCV-specific CD8+ T cells retain a chronic scar after cure of chronic HCV infection
Relations
BioSample SAMN14891028
SRA SRX8325490

Supplementary file Size Download File type/resource
GSM4546505_Pat3_cHCV_13.coutt.csv.gz 226.8 Kb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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