GSM9266462: CART22_Rep1; Homo sapiens; RNA-Seq
Bulk RNA-sequencing  -  Pellets were transformed to total RNA using Qiagen's QiaShredder (Cat no. 79656) and RNeasy Mini RNA extraction kit scRNA-sequencing - Cryopreserved peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear cells (BMMC) were thawed and resuspended in media. Suspensions were centrifuged at 300 x g for 5 minutes at 4°C and resuspended in Dulbecco's phosphate-buffered saline (DPBS, Gibco, PN 14190-144) with 1% bovine serum albumin (BSA, Miltenyi Biotec, PN 130-091-376). Cell suspensions were then centrifuged at 500 x g for 5 minutes at 4°C and resuspended in 50 µL of cell staining buffer (Biolegend Inc, PN 420201) followed by addition of 5 µL of Human TruStain FcX™ blocking reagent (Biolegend Inc, PN 422301) if the cell count per sample in DPBS + 1% BSA was > 200,000 cells. Cells were incubated in Fc block for 10 minutes at 4°C. During incubation, hashtag antibodies were prepared by adding 2.2 µL (1.5 µg) of each of the TotalSeq™ anti-human Hashtag to 52.8 µL cell staining buffer and centrifuged at 14000 x g for 10 minutes at 4°C. At the end of incubation in Fc block, each cell suspension was labelled with 50 µL TotalSeq™ anti-human Hashtag antibody and incubated for 30 minutes at 4°C. Cells were then washed thrice in cell staining buffer at 500 x g for 5 minutes at 4°C and resuspended in DPBS + 0.04% BSA. If cell counts in DPBS + 1% BSA were < 200, 000 cells, cells were centrifuged at 300 x g for 5 minutes at 4°C and resuspended in DPBS + 0.04% BSA. Bulk RNA-sequencing - Complementary DNA libraries were prepared with Illumina's Stranded mRNA poly-A enrichment kit following the standard protocol. Individual library QC was performed on Agilent TapeStation prior to pooling. Pooled libraries were sequenced on NextSeq2000 on a P4 XLEAP SBS flow cell to collect 20 million reads per sample. scRNA-sequencing - Hashed and pooled cell suspensions or individual cell suspensions were subsequently processed to generate single cell RNA-seq libraries using chromium Next GEM single cell 3' reagent kits v3.1 Dual Index (10x Genomics, PN-1000268) with 3' feature barcode technology (10x Genomics, PN-1000262) for cell surface protein using chromium controller as per manufacturer's instructions. Library fragment size was determined using the Bioanalyzer Agilent 2100 with the High Sensitivity DNA chip (Agilent Technologies, 5067-4626) and libraries were quantified using Kapa qPCR quantification kit (Roche Diagnostics, PN 501965234). After pooling, libraries were sequenced on an Illumina NovaSeq 6000 using sequencing parameters read1: i7: i5: read2 :: 28:10:10:90 bp at an average sequencing depth of 50,000 read pairs per cell.
GEO
GSM9266462_r1