GSM7029161: LV3628_2_h6_NPCday14_S18; Homo sapiens; RNA-Seq
fbNPCs: iPSCs were differentiated into forebrain neural progenitors (fbNPCs) as previously described Johansson et al., 2022 and Grassi et al., 2020. Upon dissociation at 70-90% confluency, the cells were plated on LN111 (1.14µg/cm2; Biolamina) coated Nunc multidishes at a density of 10.000 cells/cm2 and grown in N2 medium (1:1 DMEM/F-12 (21331020; GIBCO) and Neurobasal (21103049; GIBCO) supplemented with 1% N2 (GIBCO), 2 mM L-glutamine (GIBCO), and 0.2% penicillin/streptomycin). 10 μM SB431542 (Axon) and 100 ng/ml noggin (Miltenyi) were given for dual SMAD inhibition. The media was changed every 2-3 days. On day 9, N2 media without dual SMAD inhibitors were used. On day 11, cells were dissociated and replated on LN111 coated Nunc multidishes at a density of 800.000 cells/cm2 in B27 medium (Neurobasal supplemented with 1% B27 without vitamin A (GIBCO), 2 mM L-glutamine and 0.2% penicillin/streptomycin Y27632 (10 μM), BDNF (20 ng/ml; R&D), and L-ascorbic acid (0.2 mM; Sigma). Cells were kept in the same media until day 14 when cells were harvested for downstream analysis. Organoids: To generate the human cerebral-like organoids we followed the protocol detailed in Johansson et al., 2022. We used three hIPSC6-derived lines obtained by transduction and FACS sorting as described above: one control line (guide against LacZ) and two LINC01876 CRISPRi KD lines (guide 2 and guide 3). Briefly, 8000 cells/well were plated in a 96-wells plate (Costar, Ultra Low Attachment, round bottom; REF 7007) with 250 μL of mTeSR1 (StemCell Technologies, Inc.) and RY27632 10 μ