title: iPS2-10X-seq Neural organoid differentiation
library_construction_protocol: Human neural organoids were cultured for 30 days following the protocol outlined by Velasco et al. (2019) PMID: 31168097. iPS2seq transfection pools were differentiated separately, and tetracycline was added to the treated organoids throughout the culture. Organoids were dissociated into single-cell suspensions using papain-based enzymatic digestion as described in Protocol Exchange (https://protocolexchange.researchsquare.com/article/pex-258/v1). Cell counts and live cell estimation were determined using a hemocytometer, and suspensions were adjusted to 2 million cells per tube. Then cells were labeled with CMO, sorted for vital cells and then loaded on the 10X controller. Cell Multiplexing Oligo (CMO) labeling was performed according to the manufacturer's protocol (CG000391) with an additional washing step. Cells were pooled in equal quantities, and the cell-pooled suspension was counted to ensure a final concentration of 1,600 cells/μL to obtain a final cell recovery of 30000 cells. Single-cell libraries were prepared using the Chromium Next GEM Single Cell 3' v3.1 protocol (CG000388 Rev C). Cells were loaded on a chip G. CG000388 Rev B (with cell multiplexing oligos). hPS-2seq barcode enrichment protocol was performed with custom primers binding on the TET repressor.