GSM7957064: M0-FPCO; Homo sapiens; RNA-Seq
M0-FPCO at day14 was digested into single cell suspension with collagenase (Sigma-Aldrich) and Pronase (Roche) in reaction buffer. The obtained cell suspension were filtered 70-μm cell straner (SPL), and re-suspended in PBS(-) to avoid cell-cell attachment. Chromium NEXT GEM Single Cell 3' Kit v3.1 (PN-1000130, 10X Genomics), Chromium NEXT GEM Chip G Single Cell Kit (PN- 1000127) and Chromium NEXT GEM Single Cell 3' v3.1 Gel Beads (PN-1000129, 10X Genomics) were used to create the library according to Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) USER GUIDE. 5000 cells in FPCO-derived single cell suspension were loaded into the Chromium Controller to create single-cell Gel-Beads emulsions (GEMs). GEMs were subjected to reverse transcription in a T100 TM Thermal Cycler (Biolad). The resulting cDNA products were recovered from the GEMs using Recovery Agent and cleaned up using Dynabeads MyONE Saline (Beckman Coulter). The cDNA products were then subjected to amplification in a T100TM Thermal Cycler according to the manufacturer's protocol. The amplified cDNA products were cleaned up by SPRIselect (Beckman Coulter) and then their concentrations were measured using a high sensitivity D5000 ScreenTape (Agilent technology). The cDNA products were then fragmented by enzyme, end-repaired and poly A-tailed, and cleaned up again by SPRIselect. Next, adaptor ligation and subsequent cleanup by SPRIselect were performed, Only appropriately labeled samples were obtained by double-size selection using SPRIslect. Samples were quality-checked using a high sensitivity D5000 ScreenTape (Agilent technology) and sequenced on a NovaSeq6000 (Illumina) at pair-end 150 bp.