GSM5641679: pancreatic lymphnode CD8+NRP-V7+ [pLN_4]; Mus musculus; OTHER
NRP-V7+ CD8+ T cells were isolated from the pancreatic lymph node or pancreas via FACS and sorted into PBS + 0.04% BSA for analysis of cell count and viability. Single-cell RNA-Seq (chemistry: Single Cell 5' R2-only: Sorted cells were stained with Trypan blue and Countess II Automated Cell Counter (ThermoFisher) was used to assess both cell number and viability. Following QC, the single cell suspension was loaded onto Chromium Chip A (10X Genomics PN 230027) and GEM generation, cDNA synthesis, cDNA amplification, and library preparation of 1,700-6,500 cells proceeded using the Chromium Single Cell 5' Reagent Kit (10X Genomics PN 1000006) according to the manufacturer's protocol. cDNA amplification included 14-18 cycles and 9-48ng of the material were used to prepare sequencing libraries with 14-16 cycles of PCR. Indexed libraries were pooled by molarity and sequenced on a NovaSeq 6000 in a PE28/91 run using the NovaSeq 6000 S1 Reagent Kit (100 cycles) (Illumina). An average of 97 million paired reads was generated per sample. Single-cell V(D)J analysis from RNA: An aliquot of cDNA generated using the methods described above was used to enrich for V(D)J regions using the Chromium Single Cell V(D)J Enrichment Kit, Mouse T Cell (10X Genomics PN 1000071) according to the manufacturer's protocol with 10 cycles of PCR during enrichment and 9 cycles during library preparation. Indexed libraries were pooled by molarity and sequenced on a NovaSeq 6000 in a PE150 run using the NovaSeq 6000 S4 Reagent Kit (300 cycles) (Illumina). An average of 24 million paired reads was generated per sample. Cell surface protein feature barcode analysis: Amplification products generated using the methods described above included both cDNA and feature barcodes tagged with cell barcodes and unique molecular identifiers. Smaller feature barcode fragments were separated from longer amplified cDNA using a 0.6X cleanup using aMPure XP beads (Beckman Coulter catalog # A63882).  Libraries were constructed using the Chromium Single Cell 5' Feature Barcode Library Kit (10X Genomics PN 1000080) according to the manufacturer's protocol with 9 cycles of PCR. Indexed libraries were pooled by molarity and sequenced on a NovaSeq 6000 in a PE28/91 run using the NovaSeq 6000 SP, S1, or S2 Reagent Kit (100 cycles) (Illumina). An average of 23 million paired reads was generated per sample.
GEO Accession
GSM5641679