MAGE-TAB Version 1.1 Investigation Title Transcriptomic profiling of antigen-specific T cells at the single-cell level after clearance of hepatitis C virus Experimental Design pathogenicity design stimulus or stress design Experimental Design Term Source REF EFO EFO Experimental Design Term Accession Number EFO_0001761 EFO_0001762 Experimental Factor Name stimulus cell line single cell identifier Experimental Factor Type stimulus cell line single cell identifier Experimental Factor Term Source REF EFO Experimental Factor Term Accession Number Person Last Name Luciani Rizzetto Eltahla Person First Name Fabio Simone Auda Person Mid Initials A Person Email luciani@unsw.edu.au s.rizzetto@unsw.edu.au Person Phone Person Fax Person Affiliation Systems Medicine in Infectious Diseases, Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney, Australia Systems Medicine in Infectious Diseases, Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney, Australia Systems Medicine in Infectious Diseases, Infl ammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney, Australia Person Address Person Roles investigator data analyst;submitter experiment performer Person Roles Term Source REF Person Roles Term Accession Number Quality Control Type Quality Control Term Source REF Quality Control Term Accession Number Replicate Type Replicate Term Source REF Replicate Term Accession Number Normalization Type Normalization Term Source REF Normalization Term Accession Number Date of Experiment 2015-06-26 Public Release Date 2017-01-31 PubMed ID 26860370 Publication DOI 10.1038/icb.2016.16 Publication Author List Auda A Eltahla, Simone Rizzetto, Mehdi R Pirozyan, Brigid D Betz-Stablein, Vanessa Venturi, Katherine Kedzierska, Andrew R Lloyd, Rowena A Bull and Fabio Luciani Publication Title Linking the T cell receptor to the single cell transcriptome in antigen-specific human T cells Publication Status published Publication Status Term Source REF Publication Status Term Accession Number Experiment Description We used scRNA-seq data generated using Smart-seq2 to reconstruct the native TCR from Ag-specific T cells and then to link these with the gene expression profile of individual cells. antigen specific CD8 T cells were obtained from a subject who had previously cleared hepatitis C virus (HCV) infection. A cell line was also generated from the same subject and included in the analysis with and without antigen stimulation. Protocol Name P-MTAB-50510 P-MTAB-51259 P-MTAB-50511 P-MTAB-50508 Protocol Type nucleic acid library construction protocol growth protocol nucleic acid sequencing protocol sample collection protocol Protocol Term Source REF EFO EFO EFO EFO Protocol Term Accession Number EFO_0004184 EFO_0003789 EFO_0004170 EFO_0005518 Protocol Description Single cell RNA sequencing was performed using Smart-seq2 as described by Picelli et al. (Picelli et al. Nature methods 2013) with the following modifications. Cell lysis buffer was prepared by adding 1 lRNase inhibitor (Clontech, Mountain View, CA) to 19 lTriton X-100 solution (0.2% v/v). Cells were sorted directly into tube containing 2 ul lysis buffer, 1 ul dNTP mix (10mM) and 1 ul oligo-dT primer at 5 M. Tubes were stored at -80 C until needed. Reverse-transcription and PCR amplification were performed as described with the exception of reducing the IS PCR primer to a 50 nM final concentration and increasing the number of cycles to 28. Sequencing libraries were prepared using the Nextera XT Library Preparation Kit (Illumina; San Diego, CA, USA) and sequencing was performed on Illumina MiSeq and NextSeq sequencing platforms. PBMC were separated from freshly collected whole blood by density gradient centrifugation, before 4 10^5 cells per well were cultured in medium (AIM-V with 1% Glutamax, 1% Penicillin-Streptomycin) supplemented with 10% heat inactivated human AB serum (Sigma, USA) in 96-well U-bottomed plates in presence of 10 g ml 1 of the CINGVCWTV peptide (aa 10731081 of HCV non-structural 3 protein). For the initial culture, the cytokines, IL-7 (10 ng ml 1, Miltenyi Biotec), IL-12 (300 g ml 1, Miltenyi Biotec) and IL-15 (10 ng ml 1, Miltenyi Biotec) were added. Fresh AIM-V media and IL-2 (20 U ml 1, Miltenyi Biotec) was added to the wells on days 4 and 7 post-stimulation. On day 10 post-stimulation, cells were re-stimulated with irradiated autologous monocytes supplemented with IL-2, IL-7, IL-12 and IL-15 in same concentrations as stated for day 1. On day 14 and 17 fresh AIM-V media containing IL-2 (U ml 1) was added to the wells. The established cell line was harvested at day 20 and cryopreserved in RPMI with 20% DMSO. Re-stimulation of the cell line was performed with 10 g ml 1 of the CINGVCWTV peptide per well (2 x 10^5 cells in each well) for 24 h. Single cells were isolated from cell lines by flow cytometry NextSeq HCV-specific CD8+ T cells were stained with NS31073-Dextramer (Immudex; Copenhagen, Denmark). Live/Dead fixable blue dye (Invitrogen) was used to exclude non-viable cells from the analysis. Subsequently, cells were washed and surface stained with the following monoclonal antibodies (mAbs): CD3-APC- Cy7, CD8-Alexa Fluor700 and CD19-PE-Cy5 (BD Biosciences). After the final wash the cells were resuspended in 500 l of PBS, 2mM EDTA and 0.5% BSA (Sigma-Aldrich, Germany) solution kept in dark at 4 C until data acquisition. After exclusion of non-viable/CD19+ cells, CD3+ CD8+ dextramer+ cells were sorted directly into 96-well PCR plates (Eppendorf) using a BD Influx cell sorter (BD Influx; BD Biosciences, Franklin Lakes, NJ) and stored at 80 oC for subsequent TCR analysis. Protocol Parameters Protocol Hardware NextSeq 500 Protocol Software Protocol Contact Term Source Name EFO ArrayExpress Term Source File http://www.ebi.ac.uk/efo/ http://www.ebi.ac.uk/arrayexpress/ Term Source Version 2.38 SDRF File E-MTAB-4850.sdrf.txt Comment [Submitted Name] Transcriptomic profiling of antigen-specific T cells at the single-cell level after clearance of hepatitis C virus Comment [SecondaryAccession] ERP020994 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR1797277-ERR1797528 Comment [AEExperimentType] RNA-seq of coding RNA from single cells Comment[ArrayExpressAccession] E-MTAB-4850 Comment[EAExpectedClusters] 2 Comment[EAExperimentType] differential Comment[EACurator] Silvie Fexova