GSM8880148: Undifferentiated pig ESCs; Sus scrofa; RNA-Seq
The cells pretreated with 10 µM Y-27632 for 24 h were dissociated using TrypLE™ Express and filtered using a 100 µm Cell Strainer (Corning Inc., cat.CLS431752). After centrifugation at 300g for 5 min at RT and removal of the supernatant, cells were washed twice with cold Ca2+ and Mg2+-free 0.04% BSA (Sigma-Aldrich; cat.A6003)/PBS (Welgene; cat.LB 001-02) at 300 × g for 5 min at 4°C, resuspended in the same buffer, and filtered through a 40 µm Flowmi Cell Strainer (SP Bel-Art; cat.H13680-0040). Viability was assessed using acridine orange and propidium iodide staining (Logos Biosystems; cat.F23001) on a LUNA-FX7™ cell counter (Logos Biosystems). RNA was not extracted separately; poly(A)+ RNA was captured directly from single cells within GEMs using the 10x Genomics Single Cell 3' v3.1 workflow. Single-cell RNA-seq libraries were prepared using the 10x Chromium iX Controller and Next Gem Single cell 3' Kit v3.1 (10x Genomics; PN-1000268) according to the 10x Chromium Next GEM Single Cell 3' v3.1 protocol (10x Genomics; doc.CG000315). Cells were loaded per channel into a Next GEM Chip G (10x Genomics; PN-1000120) with reverse transcription reagents (PN-1000123), partitioning oil (PN-1000190), and Next GEM Single Cell 3ʹ Gel Bead (10x Genomics; PN-1000122) to generate GEMs. Barcoded cDNA was synthesized and amplified according to the Next GEM Single Cell 3' Kit v3.1 protocols (doc.CG000315). Library preparation was performed using the Single Cell 3' Library Kit v3.1 (PN-1000190), including fragmentation (Fragmentation Enzyme, PN-2000090; Fragmentation Buffer PN-2000091), end-repair/A-tailing with P5/P7 sequences, adaptor ligation (Ligation Buffer, PN-2000092; Adaptor Oligos, PN-2000094; DNA Ligase, PN-220110), and index PCR with i5/i7 sequences, according to the manufacturer's protocol (10x Genomics; doc.CG000315). Libraries were quantified using qPCR (LightCycler Instrument) according to the qPCR Quantification Protocol Guide (KAPA Biosystems) and qualified with an Agilent 4200 TapeStation (Agilent Technologies).