GSM8239742: control group, replicate 1; Homo sapiens; RNA-Seq
Fresh tumor and adjacent normal tissue samples were surgically removed from patients and immersed in a complete medium containing 90% Dulbecco's modified eagle medium (DMEM; Cat# 11054001, GIBCO) and 10% fetal bovine serum (FBS; Cat# 16140071, GIBCO), and transported to the lab in a refrigerated container. Suitable small tissue blocks (no necrotic foci / no hemorrhagic foci /less fibrous connective tissue) were cut into pieces (diameter 1-3 mm). The pieces were transferred to the gentle MACS C Tubes (Cat# 130-096-334, Miltenyi Biotec), with 5 mL of digestive enzyme included in Tumor Dissociation Kit (Cat# 130-095-929, MiltenyiBiotec). Then the tissues were made into single-cell suspension using the gentleMACS Dissociator (Cat# 130-093-235, MiltenyiBiotec) following these steps: milled; incubated at 37C for 30 min on a shaker; milled; incubated at 37C for 30 min; milled; filtered through a 70 mm filter, plus 2% FBS. Finally, the single-cell suspension was centrifugated at 400 g for 7 mins, resuspended in CELLSAVING (Cat# C40050, NCM biotech), cryopreserved in Cryotubes (Cat# 430488, Corning) and then stored at -80℃. This process is based on the microfluidic platform Chromium ™ Single cell 3'Solution. The primers were composed of full-length Illumina TruSeq Read 1 sequencing primer, 16bp 10X Barcode sequence (used to distinguish cells), 12 bp unique molecular identifier (UMI) (to distinguish different transcripts of unified cells and remove PCR Duplications), and 30 bp poly dT reverse transcription primer. The prepared cell suspension, 10X barcode gel magnetic beads and oil drops were added to different channels (8 channels in total) of Chromium Chip B respectively, and GEM was formed through the microfluidic "double cross" cross system. In order to obtain a single cell reaction system, the concentration of cell suspension was controlled at 700-1200 cells/ul, so 90-99% of GEM produced did not conThis process is based on the microfluidic platform Chromium ™ Single cell 3'Solution. The primers were composed of full-length Illumina TruSeq Read 1 sequencing primer, 16bp 10X Barcode sequence (used to distinguish cells), 12 bp unique molecular identifier (UMI) (to distinguish different transcripts of unified cells and remove PCR Duplications), and 30 bp poly dT reverse transcription primer. The prepared cell suspension, 10X barcode gel magnetic beads and oil drops were added to different channels (8 channels in total) of Chromium Chip B respectively, and GEM was formed through the microfluidic "double cross" cross system. In order to obtain a single cell reaction system, the concentration of cell suspension was controlled at 700-1200 cells/ul, so 90-99% of GEM produced did not contain cells, and most of the remaining GEM contained one cell