GSM7682692: Control group, rep 1, scRNAseq; Homo sapiens; RNA-Seq
The tumor constructs were carefully separated from the stroma and then removed and pooled together for subsequent processing. The sample suspension was digested using a tumor digestion buffer (TTDR, BD Horizon) according to the manufacturer's instructions and incubated on a rocker in a cell culture incubator for one hour with vigorous pipetting every 10 minutes. The same digestion buffer was added to the microdevices without the tumor constructs to digest the stroma gels, which were separately collected and pooled together to obtain single cell suspensions. All cell suspensions were passed through 70 µm nylon mesh cell strainers to remove undigested clusters and multiplets. The isolated cells were collected and washed with PBS, and then loaded onto a Chromium Single Cell Chip (10x Genomics) according to the manufacturer's instructions for co-encapsulation with barcoded beads. The capture of RNA transcripts in the barcoded cells and reverse transcription of cDNA were performed using the manufacturer's standard protocols. The Agilent TapeStation High Sensitivity D5000 ScreenTape was used for QC of generated cDNA. The barcoded cDNA was converted into single-cell RNA-seq libraries for sequencing using the Chromium Single Cell 3' Reagent Kit v3 (10x Genomics) according to the manufacturer's instructions. The Agilent TapeStation High Sensitivity D1000 ScreenTape was used for QC of prepared libraries for sizing (bp) and concentration. The final libraries were pooled together and sequenced by Illumina NovaSeq sequencer with a SP v1.5 FlowCell (100 cycles).