Time-resolved single-cell RNA-sequencing of Hi-Q brain organoids

To dissect the cell diversity of Hi-Q brain organoids and correlate it to the human brain, we performed single-cell RNA-sequencing (scRNA-seq). We analyzed 16,228 cells isolated from three organoids at each time point of day 60, 90, and 150.For scRNA library construction, we utilized the Chromium Single Cell 3' Reagent Kits v3. Single nucleus suspensions in 1XPBS containing 0.04% BSA (700-1200 nuclei/uL concentration) were assessed for viability, debris, and cell aggregates. To achieve single-cell resolution, the cells were delivered at a limiting dilution, ensuring that the majority (~90-99%) of generated GEMs contained no cells, while the remainder essentially contained a single cell. Given the complex composition of organoids, our target was to capture 2000-3000 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 were released and mixed with cell lysate and Master Mix. Incubation of the GEMs then facilitated the production of barcoded, full-length cDNA from poly-adenylated mRNA. Subsequently, the GEMs were broken, and the pooled fractions were recovered. Silane magnetic beads were employed to remove excess biochemical reagents and primers from the post-GEM reaction mixture. Full-length, barcoded cDNA was then amplified by PCR to generate sufficient mass for library construction.Enzymatic Fragmentation and Size Selection were employed to optimize the cDNA amplicon size before library construction. The R1 (read 1 primer sequence) was added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were incorporated during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contained the P5 and P7 primers used in Illumina bridge amplification. A Single Cell 3' Library comprised standard Illumina paired-end constructs that began and ended with P5 and P7. We allocated Illumina NovaSeq6000 flow cells to sequence with the first read of 28nt (containing the cell-specific barcode and UMI) and generate with the second read of 90nt 3' mRNA transcriptome data