GSM9277076: Tetramer-negative CD4+ T cells, TCR; Mus musculus; OTHER
For scRNA-seq experiments, spleen and lymph node cells were pooled from 8-week-old female NOD mice (n=6-10). After red blood cell lysis, cells were washed with MACS buffer, and the CD19+ B cells and CD11b+ myeloid cells were depleted using CD19 beads (Miltenyi Biotec, Cat #: 130-050-301) and CD11b beads (Miltenyi Biotec, Cat#: 130-097-142). Cells were then stained with fluorochrome-conjugated tetramers followed by magnetic enrichment using anti-PE and anti-APC beads. Surface staining was performed as described above and tetramer-positive and polyclonal cells were sorted using the BD FACS Aria II sorter (BD Biosciences). cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-16 cycles before being cleaned up using SPRI Select beads. Samples were then run on a Bioanalyzer to determine the cDNA concentration. V(D)J target enrichment (TCR) was performed on the full-length cDNA. Gene Expression, Enriched TCR, and Feature libraries were prepared according to the recommended protocols from the 10× Genomics Chromium Single Cell 5′ Reagent Kits User Guide (v2 Chemistry Dual Index) with Feature Barcoding technology for Cell Surface Protein and Immune Receptor Mapping, with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10× Genomics platform, the following kits were used: Chromium Next GEM Single Cell 5′ Kit v2, 16 rxns (Cat #: PN-1000263), Chromium Next GEMChip K Single Cell Kit, 48 rxns (Cat #: PN-1000286), Chromium Single Cell Human TCR Amplification Kit (Cat #: PN-1000252), Dual Index Kit TT Set A, 96 rxns(Cat #: PN-1000215), 5' Feature Barcode Kit, 16 rxns (Cat #: PN-1000256), and Dual Index Kit TN Set A, 96 rxns (Cat #: PN-1000250).The concentration of each library was accurately determined through qPCR using the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to achieve the desired cluster counts for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 151×10×10×151 sequencing recipe according to the manufacturer's protocol. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression library, and 5000 reads/cell for each V(D)J and Feature library.