GSM8991812: RN2 cells from mice treated with U5 CAR T, GEX (CD45.2+/CD11b+); Mus musculus; OTHER
Bone marrow mononuclear cells isolated from recipient mice were incubated with mouse Fc receptor blocker (BioLegend; 101320) and then stained with FITC anti-mouse CD45.1 (BioLegend; 110706), PE anti-mouse CD45.2 (BioLegend; 109808), PE-Cy7 anti-mouse CD3 (BioLegend; 100220), and Brilliant Violet 711 anti-mouse CD11b (BioLegend; 101242) antibodies in PBS supplemented with 0.5% BSA. RN2 cells (CD45.2+ CD11b+), donor-derived T cells (CD45.2+ CD3+), and recipient-derived WBCs (CD45.1+) cells were sorted using an Aria cytometer (BD Biosciences). 500,000 sorted cells were incubated with Fc receptor blocker (BioLegend, 101320), stained with TotalSeq-C Mouse Universal Cocktail (BioLegend, 199903) in cell staining buffer (BioLegend, 420201) for 30 min at 4°C, and subsequently washed with PBS supplemented with 0.04% BSA. Following reverse transcription and cell barcoding in droplets, emulsions were broken and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification.  Between 20,000 and 60,000 cells were targeted for each sample, and several samples were multiplexed together on one lane of 10X Chromium X, following cell hashing protocol using TotalSeqC (BioLgened). The scRNA-seq and scTCR-seq libraries were prepared using the 10x Single Cell Immune Profiling Solution Kit, according to the manufacturer's instructions. For gene expression library construction, amplified cDNA was fragmented and end-repaired, double-sided size-selected with SPRIselect beads, PCR-amplified with sample indexing primers (98 °C for 45 s; 14–16 cycles of 98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s; 72 °C for 1 min), and double-sided size-selected with SPRIselect beads. For TCR library construction, TCR transcripts were enriched from 2 μl of amplified cDNA by PCR (primer sets 1 and 2: 98 °C for 45 s; 10 cycles of 98 °C for 20 s, 67 °C for 30 s, 72 °C for 1 min; 72 °C for 1 min). Following TCR enrichment, enriched PCR product was fragmented and end-repaired, size-selected with SPRIselect beads, PCR-amplified with sample-indexing primers (98 °C for 45 s; 9 cycles of 98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s; 72 °C for 1 min), and size-selected with SPRIselect beads. Final libraries (GEX, TCR and HTO) were sequenced on Illumina NovaSeq X platform (R1 – 26 cycles, i7 – 10 cycles, i5 – 10 cycles, R2 – 90 cycles). 