scRNA-seq of brain organoids
For scRNA library construction, we made use of 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, thecells are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contains no cell, while the remainder largely contain 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 are recovered. Silane magnetic beads are used to remove leftover 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. Enzymatic Fragmentation and Size Selection are used to optimize the cDNA amplicon size prior to library construction. R1 (read 1 primer sequence) are added to the molecules during GEM incubation. P5, 47 P7, a sample index and R2 (read 2 primer sequence) are added during library construction via End Repair, A-tailing, Adaptor Ligation and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification. A Single Cell 3 Library comprises standard Illumina paired-end constructs which begin and end with P5 and P7. We allocated Illumina NovaSeq6000 flowcells to sequence with the first read 28nt (cell specific barcode and UMI) and generate with the second read 90nt 3mRNA transcriptome data.