GSM7029160: LV3628_1_h6_NPCday14_S14; 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 μM. This is considered day −5 of the differentiation of the iPSCs-derived hFB organoids. On days −3 and −1 the medium was changed (150 μL and 200 μL of mTeSR1, respectively). At day 0 the cells are fed with Neural Induction Medium (NIM; DMEM/F12 media, N2 Supplement (1:100), L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:100) and Heparin (2ug/ml).) enriched with 3% KSR. On days 2, 4, and 6, the organoids were fed with NIM with no added KSR. On day 8 the organoids were embedded in 30-50 μL of Matrigel (Corning) and incubated at 37°C for 25 minutes to allow the Matrigel to solidify. The organoids were then transferred in Corning REF 3471 6-well plates with flat bottoms containing 4  ml/well of Cortical Differentiation Medium (CDM; F12 Media (-Glut) (48.5%), Neurobasal (48.5%), N2 Supplement (1:200), B27 Supplement (-Vit.A, 1:100), L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:200), Beta MercaptoEtOH (50uM) and Insulin (2.5 ug/mL)). On days 10 and 12 of the differentiation, the media was changed exchanging 3 ml/well for 3 mL of fresh CDM. On days 15, 17, 19, 21 and 23, ∼4 mL of the medium was replaced with 4 mL of Improved Differentiation Medium + A (IDM, F12 Media (-Glut) (48.5%), Neurobasal (48.5%), N2 Supplement (1:200), B27 Supplement (+Vit.A, 1:50), L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:200), Beta MercaptoEtOH (50uM), Insulin (2.5 ug/mL) and Ascorbic Acid (400uM)). From day 25, the media was changed every 3 days with 3-4 mL of Cortical Terminal Differentiation Medium (CTDM, F12 Media (-Glut) (48.5%), Neurobasal (48.5%), N2 Supplement (1:200), B27 Supplement (+Vit.A) – (1:50) 800uL, L-Glutamine (2mM), Penicillin/Streptomycin (1:500), Non-Essential Amino acids (1:200), Beta MercaptoEtOH (50uM), Insulin (2.5 ug/mL) and Ascorbic Acid (400uM), BDNF (10ng/uL), cAMP (200uM), GDNF (10ng/uL)). All the diameter measurements of the organoids were taken with the Measure tool from Image J. The chosen measuring unit was mm. Organoids: The nuclei isolation from the organoids was performed according to Jönsson et al., 2018. In brief, the tissue and organoids were thawed and dissociated in ice-cold lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM DTT) using a 1 ml tissue douncer (Wheaton). The homogenate was carefully layered on top of a sucrose cushion (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl, pH 8.0, and 1 mM DTT) before centrifugation at 30,000 × g for 2 hours and 15 min. Pelleted nuclei were softened for 10 min in 100 ml of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) before being resuspended in 300 ml of dilution buffer (10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) and run through a cell strainer (70 mm). Cells were run through the FACS (FACS Aria, BD Biosciences) at 4°C at a low flow rate using a 100 mm nozzle (reanalysis showed >99% purity). Nuclei were directly loaded onto the Chromium Next GEM Chip G or Chromium Next GEM Chip K Single Cell Kit along with the reverse transcription mastermix following the manufacturer's protocol for the Chromium Next GEM single cell 3' kit (10X Genomics, PN-1000268) to generate single-cell gel beads in emulsion. cDNA amplification was done as per the guidelines from 10x Genomics using 13 cycles of amplification for 3' libraries. fbNPCs: Total RNA was isolated from nuclei, cell culture samples, or tissue using the RNeasy Mini Kit (Qiagen). Organoids: Sequencing libraries were generated with unique dual indices (TT set A) and pooled for sequencing on a Novaseq6000 using a 100-cycle kit and 28-10-10-90 reads. fbNPCs: Libraries were generated using Illumina TruSeq Stranded mRNA library prep kit (poly-A selection) and sequenced on a NextSeq500 (PE 2×150bp).