title: GSM6745554: Cerebral Organoid; Homo sapiens; RNA-Seq
library_construction_protocol: Organoids were transferred to individual sterile tubes and rinsed with PBS. To each organoid, 500 µL of dissociation solution (250 U/mL papain plus 2000 U/mL DNase I in Hanks' Balanced Salt Solution [HBSS]) was added. Organoids were incubated at 37°C for 30 minutes on an orbital shaker (Infors HT Celltron) set at 90 rpm. After incubation, a 1 mL pipettor was used to triturate each organoid 5 - 10 times. The cell suspension was incubated at 37°C for an additional 10 minutes on an orbital shaker set at 90 rpm. The cell suspension was triturated an additional 5 - 10 times. The dissociation reaction was quenched by adding 2 mL of ovomucoid protease inhibitor solution (10 mg/mL ovomucoid protease inhibitor in HBSS). Cells were centrifuged at 300 x g for 5 minutes and resuspended in HBSS followed by a cell count using AO/DAPI staining and analysis on NucleoCounter® NC-200™ (Chemometec). The cell suspension was passed through a 37 µm strainer to remove any additional aggregates. One million cells were labeled using TotalSeq™-B anti-human oligo-tagged antibody (Biolegend) using a concentration of 1 µg/mL for 30 minutes at room temperature. Cells were rinsed twice with sterile PBS and mixed in equal proportions to obtain > 2500 cells per organoid. Library was prepared using Chromium Single Cell 3ʹ v1 protocol with Feature Barcoding technology (10x Genomics) following surface protein staining with TotalSeq™–B (BioLegend)