GSM8677774: MN07_gRNA1_Rep2_snRNA_org_hiPS6_LV3824_L1_CRISPRi_d15_batch4; Homo sapiens; RNA-Seq
The nuclei isolation from theday 15 cerebral organoids was performed according to Jönsson et al., 2021. In brief, 5 organoids per replicate were 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 𝝻l of dilution buffer (10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) and run through a cell strainer (70 𝝻m). Nuclei were run through the FACS (FACS Aria, BD Biosciences) at 4°C at a low flow rate using a 100 𝝻m nozzle (reanalysis showed >95% purity). For the snRNAseq libraries, 8500-10000 nuclei were directly loaded onto the Chromium Next GEM Chip G  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. Sequencing libraries were generated with unique dual indices (TT set A). The generated libraries for snRNA sequencing were sequenced on a Novaseq6000 or Novaseq X plus (paired end, ~ 150 bp) using a 100-cycle kit and 28-10-10-90 reads.