title: SmartSeq single-cell RNAseq of SARS-CoV-2 spike-specific CD4+ T cells
design: SmartSeq single-cell RNAseq of SARS-CoV-2 spike-specific CD4+ T cells
library_construction_protocol: Patients were recruited from the John Radcliffe Hospital in Oxford, UK, between March 2020 and September 2021 by identification of patients hospitalized during the SARS-CoV-2 pandemic and recruited into the Sepsis Immunomics study. All patients were sampled at least 28 days after symptom onset during the primary infection, while 10 patients were further sampled around 3-4 years after initial infection. Single cells were directly sorted into 96-well PCR plates (Thermo Fisher Scientific) containing cell lysis buffer and stored at −80°C for further SmartSeq2 analysis. ScRNA-seq with ex vivo tetramer+ cells was performed using SmartSeq2 analysis as described previously (Picelli et al (2014)). Reverse-transcription (RT) and PCR amplification were performed with the exception of using ISPCR primer with biotin tagged at the 5′ end and increasing the number of cycles to 25. Sequencing libraries were prepared using the Nextera XT Library Preparation Kit