GSM6806916: Human_Somite_Organoids_Day5; Homo sapiens; RNA-Seq
Organoids were dissociated into a single-cell suspension using the Worthington Papain Dissociation System kit (Worthington Biochemical). Cells were counted on the LUNA-FX7 Automated Cell Counter (Logos Biosystems) using fluorescence detection for viability with an acridine orange/propidium iodide stain (Part No. F23011). The sample was loaded into Chip G per the user guide from 10x Genomics, and no alterations were made at any step of the protocol (Part No. CG000315). GEMs were formed targeting 10,000 cells and reverse transcription completed immediately after. The cDNA was cleaned from the GEM reagents, amplified for a total of 11 cycles and verified via TapeStation (Agilent Technologies). Amplified cDNA was diluted and ran on the 4200 TapeStation instrument using High Sensitivity D5000 tape and reagents (Part No. 5067-5592 & 5067-5593). The amplified cDNA was fragmented, end repaired, and A-tailed followed by adaptor ligation, and PCR amplification for a total of 11 cycles with each sample receiving a unique set of dual indices (Part No. 1000215). Final libraries were diluted and ran using the High Sensitivity D5000 tape and reagents (Part No. 5067-5592 & 5067-5593) on the 4200 TapeStation (Agilent Technologies). Libraries were quantified via Kapa qPCR using the Complete Universal Kit (Part No. 07960140001, Roche Sequencing Solutions) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories).