scRNA-seq CRISPR screening of human posterior brain organoids
scRNA-seq CRISPR screening of human posterior brain organoids
Five organoids were pooled and dissociated into single cell suspensions at day 30 and day 70. Cells positive for fluorescent reporters were selected using FACS and these cells were then loaded onto the 10x Chromium using the Chromium Next GEM Single Cell 5' v2 Dual Index kit (10x Genomics, PN-1000265) targeting 10,000 cells per reaction with two reactions per time point.  The final mixed pool of cells was used to generate Qian organoids following the previously described method (Qian Reference). To induce Cas9 expression and therefore create knockouts in targeted genes, 2 μg/ml doxycycline (Clontech, 631311) was added with each media change starting on day 7 until day 14. Organoids were returned to their normal media on day 15. Fluorescence was monitored throughout culturing to ensure KOs reporters were not silenced throughout differentiation. Cells positive for fluorescent reporters were selected using FACS and these cells were then loaded onto the 10x Chromium using the Chromium Next GEM Single Cell 5' v2 Dual Index kit (10x Genomics, PN-1000265) targeting 10,000 cells per reaction with two reactions per time point. Single-cell transcriptome and CRISPR gRNA libraries were prepared following manufacturer protocols. Cells positive for fluorescent reporters were selected using FACS and these cells were then loaded onto the 10x Chromium using the Chromium Next GEM Single Cell 5' v2 Dual Index kit (10x Genomics, PN-1000265) targeting 10,000 cells per reaction with two reactions per time point. Single-cell transcriptome and CRISPR gRNA libraries were prepared following manufacturer protocols.