GSM8174733: pMEL CD8 cells citeseq hashtages; Mus musculus; OTHER
Single-cell libraries were prepared from sorted live CD8+ T cells derived from either polyclonal mice or CD8+ pMEL cells from an antigen-specific system, utilizing Chromium Single Cell 5' Reagent (v1 Chemistry) or Chromium Next GEM Single Cell 5' Reagent (v2 Chemistry Dual Index) immediately post-cell isolation. Sample multiplexing was achieved via CITEseq, wherein cells were stained with TotalSeq-C anti-mouse hashtag antibodies and additional surface flow cytometry antibodies (TCRβ, CD8). Sorted cells, tagged with uniquely barcoded TotalSeq-C antibodies, were pooled and loaded into parallel lanes for droplet generation, targeting a recovery of 2,000 cells per sample. After droplet partitioning, cells underwent lysis and reverse transcription within droplets. cDNA was isolated, bulk amplified, and selected with SPRIselect beads. Adaptors were ligated, sample indices were added through PCR, and samples underwent another selection using SPRIselect beads. Library concentration was determined via PCR with KAPA DNA Quantification.