Homo sapiens:brain organoids:D150
For scRNA library construction, we used the Chromium Single Cell 3 Reagent Kits v3. Single nucleus suspensions in 1XPBS containing 0.04% BSA (700-1200 nuclei/L concentration) have been checked for viability, debris, and cell aggregates. To achieve single-cell resolution, the cells are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contain no cell. In contrast, the remainder essentially includes a single cell. Because of the complex composition of organoids, we aim to target 2.000-3.000 cells per sample. Upon dissolution of the Single Cell 3 Gel Bead in a GEM, primers containing (i) an Illumina R1 sequence (read 1 sequencing primer), (ii) a 16 bp 10x Barcode, (iii) a 12 bp Unique Molecular Identifier (UMI) and (iv) a poly-dT primer sequence are released and mixed with cell lysate and Master Mix. Incubation of the GEMs then produces barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, the GEMs are broken, and the pooled fractions recovered. Silane magnetic beads remove excess biochemical reagents and primers from the post-GEM reaction mixture. Full-length, barcoded cDNA is then amplified by PCR to generate sufficient mass for library construction.