title: GSM8485916: 232-HBO (Control hindbrain organoid); Homo sapiens; RNA-Seq
library_construction_protocol: Tissues were lysed by adding 1 mL chilled Lysis Buffer, andsuspension was filtered using a 30μm MACS® SmartStrainer to remove cell debris and large clumps and nuclei were pelleted by centrifugation at 500 rcf for 5 min and at 4°C. The supernatant was removed without disrupting the nuclei pellet and pellets were washed in 0.7ml nuclei wash and resuspension buffer  incubated on ice for 15-30 min whilst being physically disrupted by pipetting up and down gently. Then, the suspension was filtered using a 30μm MACS® SmartStrainer to remove cell debris and large clumps and nuclei were pelleted by centrifugation at 500 rcf for 5 min and at 4°C. The supernatant was removed without disrupting the nuclei pellet and pellets were washed in 0.7ml nuclei wash and resuspension buffer. Nuclei-pellets were suspended in the DAPI solution (5µl DAPI (300µM) in 1000µl 1× PBS) and incubated for 5 min prior to FACS sorting. In the subsequent FACS sorting, single DAPI-positive nuclei were filtered by size and granularity using a calibrated FACS ARIA III Cell Sorter (BD Biosciences) to minimize the amount of cell debris in the suspension, using 4-way purity sort with 32 purity mask. Based on the number of intact nuclei obtained, we adjusted the amount of nuclei suspension buffer to yield a single nuclei solution of ~1000 nuclei/μl and proceeded immediately with 10X Chromium. After nuclei extraction an individual lane per sample ofChromium Next GEM Single Cell 3' Kit v3.1 chemistry was run on a Chromium Controller device(10X Genomics). Libraries were generated following manufacturer's recommendations.