GSM6008297: Sample_02_NA; Homo sapiens; RNA-Seq
GSM6008297
Surgically resected CLM specimens were chopped into small fragments and subjected to GentleMACS (Miltenyi) dissociation in  Collagenase D solution for 35 min at 37°C. The resulting single cell suspension was filtered through a 100 µm cell strainer and erythrocytes were lysed with ACK. Cells were then stained for flow cytometry, sorted and resuspended in 0.5 ml PBS 1X plus 0.04% BSA and washed once by centrifugation at 450 rcf for 7min. Cells were then resuspended in 50 ml and counted with an automatic cell counter (Countess II, Thermo Fisher). Approximately 10.000 cells for each sample were loaded into the Chromium Chip B using the Single Cell reagent kit v3 (10X Genomics) for Gel bead Emulsion generation into the Chromium system Following capture and lysis, cDNA was synthesized and amplified for 14 cycles following the manufacturer's protocol. 50 ng of the amplified cDNA were then used for each sample to construct Illumina sequencing libraries. Sequencing was performed on the NextSeq550 Illumina sequencing platform following 10xGenomics instruction for read generation, reaching at least 35000 reads as mean reads per cell.