Agilent 4150 tape station instrument

mRNA‐isolation from a minimum of 1 × 10⁵ cells of the CD8⁺ T cell subsets was performed immediately after cell sorting using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA concentrations and the RNA integrity number (RIN) were determined with the Agilent 4150 Tape Station instrument using the Agilent High‐Sensitivity RNA Screen Tape (Agilent, Santa Clara, CA). All samples that were used for sequencing had RINe values greater than or equal to 8.8. Libraries were prepared with 14 ng RNA input per sample using the QuantSeq 3′mRNA‐Seq Library Prep Kit‐FWD and the UDI 12 nt Unique Dual Indexing Add‐on kit (both Lexogen, Vienna, Austria) according to the manufacturer's instructions. For library amplification the optimal number of cycles was assessed for each sample. The quality and concentration of the libraries was determined on an Agilent 4150 Tapestation instrument using the Agilent High Sensitivity D1000 ScreenTape (Agilent, Santa Clara, CA). The pooled cDNA libraries (libraries were diluted to 2 nM concentration) were sequenced in an Illumina NextSeq 2000 instrument (Illumina, San Diego, CA). These sequence data have been submitted to the GEO database under accession number GSE207621. The QuantSeq data analysis pipeline (Lexogen, Vienna, Austria) was used for data processing and the calculation of normalised counts.