GSM7325726: M419CD45pos; Homo sapiens; RNA-Seq
We selected two treatment-naïve high-grade ovarian serous cancer samples, one high-stromal and one low-stromal, and generated inhouse scRNA-seq data. We used FACS to sort immune cells (CD45+) and non-immune cells (CD45-) of each tumor sample. Each of the two compart was subjected to scRNA-seq as a separate library. Single-cell libraries were generated from sorted live CD45+ cells using the Chromium Single Cell 30 Reagent (5PrimeV1 and Next GEM 5PrimeV1.1 Chemistry). Sorted cells were resuspended in PBS with 0.04% bovine serum albumin (BSA; Sigma) and were counted using the Cellometer Auto2000 (Nexcelom). Cells were then loaded into the Single Cell Chip and processed through the 10X controller for droplet generation (targeting a recovery of 10,000 cells per sample), resulting in droplets containing a single cell and bead per droplet. After droplet generation, cells were subjected to in-drop lysis and reverse transcription, resulting in single-cell transcriptomes bearing bead-speciﬁc sequences to identify the cell of origin. Following reverse transcription, cDNA was isolated and ampliﬁed by 13 cycles of PCR. Ampliﬁed cDNA was then size-selected by SPRISelect Beads, and was subjected to enzymatic fragmentation, end repair, and A-tailing. Adaptors were then ligated, and sample indices were added by PCR. Samples were then size-selected by SPRISelect, and the concentration was determined by KAPA DNA Quantiﬁcation. The end result of this process is library constructs containing P5 and P7 Illumina sequencing adaptors, a 16 base pair cell barcode, 10 base pair unique molecular identiﬁer (UMI), a gene insert and an i7 sample index.