title: GSM6634347: CO_30DIV_ARX_2; Homo sapiens; RNA-Seq
library_name: GSM6634347
library_construction_protocol: For single cell dissociation and RNA seq, cortical and ganglionic eminence organoid were dissociated using Worthington papain dissociation kit (Cat. No. LK003150) as per instruction manual. Each developmental timepoint (30 DIV and 120 DIV) and each subtype of spheroid (cortical and ganglionic eminence) was processed separately. A total of 3 lines, and one clone per line, were used for each condition. Briefly, 3-4 organoids for each timepoint and subtype were collected in a small petri dish with 5 ml of prewarmed Worthington papain solution and 250 µl of Worthington DNase solution. The spheroids were chopped into small pieces using sterile blades and the dishes were placed on a digital rocker in a cell culture incubator for 60 minutes at 37 °C. Digested tissue was collected in a 15-ml tube and 5 ml of room temperature (RT) Earle's Balanced Salt Solution (EBSS) was added. The mixture was then triturated by gently pipetting up and down 10 times with 10-ml plastic pipette followed by 1-ml and 200-µl pipettes. Any undissociated tissue was allowed to settle at the bottom of the tube and the cloudy suspension was carefully aspirated in a clean 15-ml tube. Next, 3.15 ml of inhibitor solution (2.7 ml of EBSS, 300 µl of reconstituted Worthington albumin-ovomucoid solution and 150 µl of DNase solution) was carefully layered over the cloudy suspension and tubes were centrifuged at 300 g for 5 minutes at RT. The supernatant was carefully discarded, and cell pellet was gently suspended in 500 µl of cold neurobasal A media. Cell viability was determined using trypan blue. 10x Genomics single cell libraries were generated by the University of Texas at San Antonio (UTSA) Genomics Core. Cell suspensions were loaded into 10x Genomics microfluidics chips from Chip B Single Cell kit (Cat. No. 1000153) and onto the 10x Chromium controller to capture single cells in Gel Beads-in-emulsion (GEMs). 7,000 cells/sample were targeted for capture in GEMs preceding library prep following manufacturer's recommendations for 10x Genomics 3' Gene Expression Reagent kit (Cat. No. 1000269) with v3 chemistry. Sequencing was performed on either NextSeq 500 at University of Texas Health Science Center (UTHSC) at San Antonio or NovaSeq 6000 at North Texas Genome Center (NTGC) to generate 50,000 reads/cell in each sample.