GSM8153071: APRIL CAR T cells +/- IL-18 replicate 1, HTO; Mus musculus; RNA-Seq
Cells were then labeled with HTO barcodes and sorted for CD4+ or CD8+ T-cells with CD138+ MOPC315.BM excluded. Post-sort CD4 and CD8 T-cells were then re-pooled at a 1:1 ratio. n=2 biological replicates per group, ~30,000 cells per biological replicate were submitted for sequencing. Single-cell RNA sequencing of FACS-sorted cell suspensions was performed on the Chromium instrument (10X genomics) following the user guide manual for 3′ v3.1. In brief, FACS- sorted cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA to a final concentration of 1,700–2,000 cells per μl. The viability of cells was ~70%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured indroplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken, and cDNA was purified using Dynabeads MyOne SILANE followed by PCR amplification as per manual instruction. About 30,000 cells were targeted for each sample. Samples were multiplexed together on one lane of 10x Chromium (using HTO) following a previously published protocol3. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles) at a depth of ~575 million reads per sample.