GSM8208818: scRNA-seq_day90-CerebralOrganoid_HC-2; Homo sapiens; RNA-Seq
After washing with PBS, eight organoids were cut in small pieces. Then, papain and DNAse I (Worthington) were mixed in DMEM/F-12, added to the organoids and then this mix was put on an orbital shaker at 50 rpm to incubate for 25 minutes in total at 37⁰C. Every 10 minutes, the cell suspension was vigorously pipetted up and down. Afterwards, the reaction was quenched with DMEM/F-12 supplemented with 2% FBS. Doublets and clumps were removed with cell strainers: first with those containing 100 µm pores and then 70 µm pores. Finally, the cell suspension was centrifuged for 5 minutes at 300 rcf and resuspended in 250 microlitre mix of DMEM/F-12 with 40% FBS, counted and checked for cell viability, and then frozen in DMEM/F-12 with 40% FBS and 15% DMSO. Cells were sequenced by Single Cell Discoveries (Utrecht, The Netherlands) according the 10X Genomics single cell 3' gel bead kit version 3 (Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3). In short, cell suspensions were thawed, the cells were washed and filtered one more time to ensure single cells. Cells were put on a Chromium single cell 3' chip, where single cells were linked to a Single cell 3' v3 gel bead and were separated from other cells by oil. Then, beads were dissolved, primers were released and the cell was lysed. The beads were coated with strands containing an Illumina TruSeq read 1, a 16 nucleotide (nt) 10X barcode, a 12 nt unique molecular identifier (UMI) and a 30 nt poly(dT)VN tail. The poly-A tails of the mRNA molecules aligned with the poly-T tails. The barcode diversity was 3.5 million. There was a different barcode per cell and a different UMI per mRNA molecule. The DNA primer was elongated to match the mRNA molecule by reverse transcription followed by template switch oligo priming and mRNA transcript extension. Then, single cell partitions were pooled again. Silane magnetic beads were used to extract the first-strand cDNA and the barcoded cDNA was amplified by PCR to complete the library preparation. P5, P7, a sample index, and TruSeq Read 2 (read 2 primer sequence) were added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contained the P5 and P7 primers used in Illumina bridge amplification. The 16 bp 10x Barcode and 12 bp UMI were encoded in Read 1, while Read 2 was used to sequence the cDNA fragment. Sequencing was performed on Illumina Nextseq 2000. Sequencing depth was set at 50,000 reads per cell for 5,000 cells per sample.