GSM5726836: Human spinal cord organoids; Homo sapiens; RNA-Seq
The hSCOs were collected in a petri dish on day 100 and chopped into small pieces. After dissection, the hSCOs were dissociated by using papain containing L-cysteine by incubation at 37℃ with gentle shaking. Papain was removed after 30 min, and the dissociated cells were washed twice with ice-cold HBSS. Libraries were prepared using the Chromium controller according to the 10x Single Cell 3' v3 protocol (10x Genomics) The dissociated cell suspensions were diluted in nuclease-free water to achieve a targeted cell count of 10,000. The cell suspension was mixed with a master mix and loaded with Single-Cell 3′ Gel Beads and Partitioning Oil into a Single Cell 3′ Chip. RNA transcripts from single cells were uniquely barcoded and reverse-transcribed within droplets. cDNA molecules were pooled. The cDNA pool was subjected to an end repair process, followed by the addition of a single 'A' base and ligation of the adapters. The products are then purified and enriched with PCR to develop the final cDNA library. The purified libraries were quantified using qPCR based on the qPCR Quantification Protocol Guide (KAPA) and qualified using the Agilent Technologies 4200 TapeStation (Agilent technologies). The libraries were subsequently sequenced using the HiSeq platform (Illumina) with a read length of 28 bp for read 1 (cell barcode and unique molecular identifier, UMI), 8 bp index read (sample barcode), and 91 bp for read 2 (RNA read). single cell RNA-seq