Illumina NovaSeq 6000 paired end sequencing; scRNA-seq of early brain organoid development from pluripotency
scRNA-seq of early brain organoid development from pluripotency
We collected these single cell suspensions from an EB time point (day 4), the time points of neuronal induction (day 7, 9 and 11) and after embedding in matrigel and starting the neuronal differentiation process (day 12, 16, 18, 21, 26, 31 and 61). Multiple organoids of each line were pooled together to obtain a sufficient number of cells. For time points just after matrigel embedding, matrigel was dissolved in Cell Recovery Solution (Corning, 354253)​​ for 15 min at 4°C. Organoids were cut in halves and washed three times with HBSS without Ca2+/Mg2+ (STEMCELL technologies, 37250). Single cell suspensions were acquired by dissociation of the organoids with a papain-based dissociation (Miltenyi Biotec, 130-092-628). Each sample was divided into a sample to be processed for scRNAseq and a sample for scATACseq, cryopreserved in Cryostor CS10, moved to -80C in a MrFrosty before moving to liquid nitrogen storage the next day. They were kept in liquid nitrogen until processesing. The cryopreserved single cell suspensions of each time point were thawed by warming up the cryo for 1-2 minutes in a water bath at 37℃ and directly centrifuged in 10ml prewarmed DMEM with 10% FBS. Cells were washed twice with PBS + 5% BSA and filtered through a 40µm cell strainer (Flomi). Cerebral organoids were generated from four different stem cell lines (H9, 409B2, Wibj2, Hoik1) simultaneously using a whole brain organoid differentiation protocol (Lancaster et al. 2013; https://doi.org/10.1038/nature12517). Single cell RNAseq libraries were generated with the Chromium Single Cell 3' V3 Library & Gel Bead Kit. Single cell encapsulation and library preparation were performed according to the manufacturer's protocol. Single cell RNAseq libraries were generated with the Chromium Single Cell 3' V3 Library & Gel Bead Kit. Single cell encapsulation and library preparation were performed according to the manufacturer's protocol.