Rna seq kit v2

CD4⁺ T cells were isolated from LNs of CD4-Cre (n=4) and Pou2af1^fl/fl x CD4-Cre (n=9) mice after immunization with SRBCs by magnetic separation using CD4⁺ T cell isolation kit (Milteny Biotec). Purity of isolated CD4⁺ T cells was ≥ 96% for all samples and is shown in Figure S14. Extraction and purification of RNA were performed using RNeasy Mini Kit (Qiagen). RNA was eluted in RNase-free water and stored at -80°C. RNA quantity was measured using the Infinite 200 Pro (Tecan).
Quality was checked using an Agilent Tape Station and all samples displayed RIN values above 9. After quantity measurement by Qubit Fluorometer (Invitrogen) 200ng of RNA were used to generate sequencing libraries using the Illumina RNA-Seq Kit V2. Sequencing libraries were subsequently quality checked on D1000 screentapes (Agilent) and 10 samples each were sequenced using a NextSeq550 (Illumina) and 2 NextSeq 500/550 High Output Kits v2.5 (75 Cycles) at the Genomics Core Facility (Ulm University).

RNA-Seq was employed to profile and quantify the entire complement of RNA molecules present in the HNSCC samples. Total RNA was extracted from the spheroids generated from UDSCC1, -5, and -6 cells on ULA surface plates. After their quality was checked using Agilent Tape Station and their quantity measured by the Qubit Fluorometer (Invitrogen, Waltham, MA, USA), 200 ng of each RNA was used to generate sequencing libraries using the Illumina RNA-Seq Kit V2. Sequencing libraries were quality checked on D1000 screen tapes (Agilent Technologies, Santa Clara, CA, USA), and samples were sequenced using a NextSeq550 (Illumina, San Diego, CA, USA) and 2 NextSeq 500/550 High Output Kits v2.5 (75 Cycles) at the Genomics Core Facility (Ulm University). High-quality reads were mapped to the human genome (hg38) using STAR (2.0.9). This was followed by the removal of multimapping reads and the conversion to gene-specific read counts for annotated genes using featureCounts (2.0.0). Data analysis was performed in R (4.0.2) using the IDE Rstudio (1.3.1056). Clustering in the heatmaps was performed using hierarchical clustering within the heatmap (1.0.12) package.