title: NextSeq 500 paired end sequencing; Transcriptomic profiling of antigen-specific T cells at the single-cell level after clearance of hepatitis C virus
design: Transcriptomic profiling of antigen-specific T cells at the single-cell level after clearance of hepatitis C virus
library_construction_protocol: 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 μl 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 °C for subsequent TCRαβ analysis. 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 μlRNase inhibitor (Clontech, Mountain View, CA) to 19 μlTriton X-100 solution (0.2% v/v). Cells were sorted directly into tube containing 2 μl lysis buffer, 1 μl dNTP mix (10mM) and 1 μl oligo-dT primer at 5 μM. 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.
attributes.tag: Experimental Factor: none
attributes.value: stimulus
attributes.tag: Experimental Factor: PB-T cell 16
attributes.value: single cell ID