Illumina HiSeq 4000 paired end sequencing; Multiome experiment of the neuroepithelial stage of brain organoid developement
Multiome experiment of the neuroepithelial stage of brain organoid developement
Single cell suspensions were acquired by dissociation of the organoids with a papain-based dissociation (Miltenyi Biotec, 130-092-628). For 19 days of development, cerebral organoids were generated from five different stem cell lines: three human induced pluripotent stem cell lines 201B7 (B7), GM25256 (WTC), HPSI0214i-kucg_2 (Kucg2), and two human embryonic stem cell line H1-hESC (H1), HES-3 simultaneously. The genotype was PAX6YFP for H1 cells, and NKX2.1GFP for HES-3 cells. For 15 days of development, cerebral organoids were generated from five human induced pluripotent stem cell lines 201B7 (B7), GM25256 (WTC), 409B2, HPSI0314i-hoik_1 (Hoik1), HPSI0214i-wibj_2 (Wibj2). Cerebral organoids were generated using a whole brain organoid differentiation protocol (Lancaster et al. 2013). For scATAC-seq, nuclei were isolated according to the protocol provided by 10x genomics (Demonstrated protocol CG000169 Rev D) using the low input protocol and a lysis time of 3 min. Nuclei were loaded in a concentration that would result in a recovery of 10,000 nuclei. In case of less nuclei recovered, the maximum number of nuclei was targeted. For sc-RNAseq, cells were put in a concentration after counting and viability checking that allowed targeting 10,000 cells and in case the cell number was not sufficient all cells were loaded. After dissociation, cell viability was checked, cells were counted and 7000 cells were targeted per lane of the 10x microfluidic chip. Single cell ATACseq libraries were generated with the Chromium Single Cell ATAC V1 Library & Gel Bead Kit. Single cell RNAseq libraries were generated with the Chromium Single Cell 3' V3 Library & Gel Bead Kit and sequenced on Illumina's HiSeq platform.