title: GSM9087547: Cortical organoids, isogenic set 1, 3 Month, Batch 3; Homo sapiens; RNA-Seq
library_construction_protocol: Individual COs of ~7 weeks and 3 months of age were washed 4x in PBS and then dissociated with a Worthington Papain Dissociation kit (Worthington Biochemical Corp.) following a modified manufacturer's protocol. In brief, live organoids were placed in an Ultra-Low Attachment 6-well plate, manually minced in a Papain+DNase solution in Earle's medium, and incubated at 37 °C in an orbital shaker at 100 RPM for 30 minutes. Then, the pieces were gently broken up with a 1 mL pipette before returning to shaking in an incubator for an additional 20-30 minutes. Afterwards, using a 10 mL pipette, the solution with the minced-up pieces was gently mixed up and down 10 times and transferred to an empty 15 mL conical tube to allow undissociated debris to settle for ~2 minutes. Subsequently, the cell suspension was transferred to a 3:8-diluted ovomucoid protease inhibitor solution in Earle's medium, mixed, and then centrifuged at 300 x g for 7 minutes. The supernatant was then discarded, and the cells were gently resuspended in 1% BSA-supplemented basal medium before filtering through a 40 µm mesh filter. Cells were then counted and confirmed to have viabilities above 85% using a Countess II Cell Counter (Thermo-Fisher). Using the Chromium Next GEM single cell 3' Cellplex v3.1 and Chip G kits (10x Genomics), the single cell partitioning, cDNA preparation, and sequencing libraries were prepared according to the manufacturer's protocol, using one 10x reaction for each experiment. After labeling the samples and amplifying the transcriptomic libraries, they were sequenced using on an Illumina Novaseq 6000 (SP or S1, depending on the run size). An average depth of ~30,000 reads per cell was targeted.
identifiers.namespace: GEO
identifiers.id: GSM9087547_r1