title: GSM8975431: Gastruloid with 2.5 days of differentiation, wild type; Mus musculus; RNA-Seq
library_construction_protocol: Gastruloids were treated with 0.5ml of TrypLE Express dissociation reagent (Life Technologies) in a 15ml conical tube at 37 °C for 5 minutes. After the initial incubation, the cell suspension was gently pipetted up and down four times and incubated for an additional 1 minute at 37 °C. To quench, 1 mL of heat-inactivated FBS was added, and the suspension was triturated gently three times to mix. Cells were pelleted by centrifugation at 900 rpm for 5 minutes at 12 °C in a tabletop centrifuge. The pellet was washed once with 1 mL of cold PBS containing 0.04% bovine serum albumin (BSA), followed by centrifugation under the same conditions. After removing the supernatant, approximately 50 µL was left to avoid disturbing the pellet. Cells were then resuspended in 500 µL of cold PBS with 0.04% BSA and filtered through a 40-μm Flowmi tip strainer (ThermoFisher Scientific, 136800040) into a pre-chilled 15 mL conical tube. A typical recovery volume was ~495 µL. Cell concentration and viability were determined, and remaining cells were kept on ice for immediate downstream processing. Two 10x genomics Single cell 3'ki V3.1 libraries were sequenced on two NovaSeq X plus runs. Bothe samples have sequencing yields of more than 406 million read per sample. The sequencing run was setup as 28 cycles+90 cycles non-symmetric run. Demultiplexing was done allowing 1 mismatch in the barcodes. Sequencing quality is good, over 97.5% of bases in the barcode regions have Q30 or above and at least 97.6% of bases in the read have Q30 or above. More than 98.2% of bases in the UMI have Q30 or above. The analysis was performed with the Cell Ranger 8.0.1 software using the default parameters. For day 2.5 Wildtype (WT) and Sp5/8dko, the number of cells captured are 9,238 and 11,190; mean reads per cell are 50,959 and 50,334, median genes per cell are 5,333 and 5,398; total number of genes are 25,523 and 25,786 respectively. Cells with extremely low number of UMI counts were filtered out.