GSM7912903: wt mouse, scRNAseq, multiplexed; Mus musculus; RNA-Seq
CD45.2+ Lin-Kit+ cells were sorted by 3-color staining on a FACS Aria sorter (BD). Samples were hashed using TotalSeqB Cell Hashing Kit and then were pooled with 3 replicates in one smaple. Approximately 15,000 pooled cells were loaded onto the 10X for each condition (control vs. heterozygous) The scRNA-Seq assay was performed according to the manufacturer's instructions (Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for Cell Surface Protein, 10x Genomics). Briefly, Total-Seq B antibody-labeled cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate a gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every GEM was reverse transcribed into cDNA, adding an Illumina TruSeq R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The DNA conjugated to the Total-Seq B antibodies (Feature Barcodes aka FBs) was also barcoded by adding an Illumina Nextera R1, UMI, and the 10x Barcode. The cell barcoded molecules were then cleaned up with Silane DynaBeads and amplified using 14 PCR cycles. Size selection using SPRIselect reagent was performed post amplification to separate full-length cDNA from FBs. Next, full-length, barcoded cDNA was then enzymatically fragmented, sized-selected, adapter-ligated, and amplified for library construction. During the library construction, P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 were added. Separately, FBs were prepared into library constructs by incorporating P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 via PCR. Samples were pooled and run on the NovaSeq 6000 sequencer with a S4 flow cell using the following sequencing parameters: R1: 28 cycles, i7: 10 cycles, i5: 10 cycles, R2: 90 cycles.