GSM8693484: PBMC6 naïve, GEX; Homo sapiens; RNA-Seq
The sample concentration and viability were assessed using the LunaFX7 fluorescence cell counter and diluted as necessary with PBS + 0.04% BSA. The cellular samples were loaded in the lanes according to the 10X Genomics Chromium Next GEM Single Cell 5'v3 (Dual Index) User Guide with a single capture lane per sample targeting a recovery of 12,000 cells per lane. Cell partitioning completed successfully with uniform emulsion consistency and the reverse transcription PCR was run overnight. All subsequent steps of library preparation, TCR enrichment, and quality control were performed as described in 10X Genomics 5' v3 Single Cell User Guide. Libraries were prepared using the 10X validated automation platform, Chromium Connect. PBMCs from PBMC6 and PBMC12 were included in the single-cell studies. For peptide-naïve PBMCs, PBMC6 and PBMC12 were thawed from frozen and incubated at a high density (1x10^7 /mL) one day before single-cell capture. For peptide-activated PBMCs, 5x10^6 PBMCs underwent weekly peptide stimulation as described earlier, with a slight difference in the duration of stimulation (6 weeks for PBMC12 and 5 weeks for PBMC6). Three days after the final peptide stimulation, the PBMCs were thoroughly mixed, and a portion of the cells was harvested for single-cell studies. The scRNA and scTCRseq libraries were sequenced two runs on a NovaSeq X-plus system, using read lengths of 28 bp for read 1, 10 bp for index 1, 10 bp for index 2, and 90 bp for read 2.