GSM8693601: hPSCs, Matrigel; Homo sapiens; RNA-Seq
Samples were washed with PBS to ensure little to no Matrigel is left, then dissociated into single cells using an incubation period of 3 min in 1 mL TrypLE Express in every well. The 1 mL solutions were neutralized with 9 mL of DMEM medium supplemented with 20% FBS, the control and Matrigel conditions were pooled, then centrifuged for 5 min at 300 RCF. After decanting the supernatant, the cell pellets were resuspended in 200 μL of E8-Flex medium and put on ice. Library preparations for the scRNAseq was performed using 10X Genomics Chromium Single Cell 3' Kit, v3 (10X Genomics, Pleasanton, CA, USA). The cell count and the viability of the samples were assessed using LUNA dual fluorescence cell counter (Logos Biosystems) and a targeted cell recovery of 10,000 cells was aimed for all the samples. Post cell count and QC, the samples were immediately loaded onto the HyDrop Hybrid38 platform. Single-cell RNA-seq libraries were prepared using manufacturers recommendations (Single-cell 3' reagent kits v3 user guide; CG00052 Rev B), and at the different check points, the library quality was assessed using Qubit (ThermoFisher) and Bioanalyzer (Agilent). 10X libraries were sequenced on MGISEQ-2000 sequencing platform and was sequenced at the MGI Hong Kong SAR sequencing facility. Before being able to sequence on the MGI platforms, the 10X libraries need to undergo a conversion step using MGIEasy Universal DNA Library Prep kit. Briefly, the final 10X sequencing libraires were circularized using the splint-ligation step and the circularized libraries were converted to single stranded DNA copies. DNA nanoballs were prepared from the circularized ssDNA using Rolling Circle Amplification (RCA). DNB libraries generated were then flown through the patterned flow cell of the MGISEQ-2000RS High-Throughput Sequencing kit. For a targeted sequencing saturation of 50-60%, sequencing were performed at a depth of 30,000 - 60,000 reads per cell. A custom sequencing recipe of paired end read of 28 bps (read 1), 100 bps (read 2) and 10 bps (index 1) was used for sequencing.