title: GSM7598126: WA01 TH #2 HESC, TH high; Homo sapiens; RNA-Seq
library_construction_protocol: hypothalamus organoids were placed in a well of a 6-well plate and washed with 5 ml PBS. The organoids were then incubated in 5 ml papain (Worthington Biochemical Corporation #LK003176) in HBSS-H (STEMCELL Technologies #37150) supplemented with DNase (Worthington Biochemical Corporation #LK003170) and 10 μM Y-27632 ROCK inhibitor for 20-40 min (depending on organoid size) on a shaker in the incubator. The organoids were gently triturated using fire-polished glass pipets of decreasing tip sizes. The cell suspension was filtered using a 70 μm strainer (Fisher Scientific #22-363-548) into a 50-ml tube containing 10 ml Hibernate-A medium (BrainBits #HA) supplemented with 1x B27 (minus vitamin A), 1% BSA solution, DNase, and 10 μM Y-27632 ROCK inhibitor. The cells were centrifuged at 800 g for 5 min and the pellet was resuspended in media. The suspension was filtered through a 35 μm strainer Barcoded cDNA was extracted from the GEMs by Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly A-tailing, adapter ligation, and 10x-specific sample indexing following the manufacturer's protocol. Libraries were quantified using Bioanalyzer (Agilent) and QuBit (Thermo Fisher Scientific) analysis and were sequenced in paired end mode on a NovaSeq instrument (Illumina) targeting a depth of 50,000 - 100,000 reads per cell.