title: GSM5555382: snRNA-Pitx2-PV-WT; Mus musculus; RNA-Seq
library_construction_protocol: Tissues were minced with sharp scissors in 250μl of HB++ (HB: 0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH, pH 7.8; ++: 0.15mM spermine, 0.5mM spermidine, 1mM DTT) and centrifuged at 500g for 5 minutes to remove excess red blood cells and fat and replaced with 250μl of fresh HB++. Sample was transferred to a 2mL Dounce homogenizer and the tube washed with an additional 250μl of HB++ to collect any remaining sample and transferred. Tissue was homogenized 30 times with loose and tight pestle before adding 32μl of 5% NP40 (in HB) and Dounced with tight pestle an additional 75 times. Sample was strained through a 40μM strainer and washed with 9.5mL of NWB (PBS with 1% BSA and 0.2U RNAse inhibitor) and spun down at 500g at 4°C for 5 minutes. The supernatant removed and nuclei resuspended in 500μl of NWB and 900μl of SCB (90% Nuclei PURE 2M Sucrose Cushion Solution and 10% Nuclei PURE Sucrose Cushion Solution; Sigma NUC-201: S9308 and S9058). The 1400μl of nuclei suspension is layered on top of 500μl SCB in a 2mL LoBind Eppendorf tube and centrifuged at 13,000g at 4°C for 45 minutes. The supernatant removed leaving ~50μl of nuclei pellet, which is resuspended in 1mL of NWB. Pellet nuclei at 500g at 4°C for 5 minutes, remove supernatant leaving ~50μl of nuclei pellet. The snRNA-seq libraries were generated using the 10x Chromium Single Cell 3' v3 reagent kit and in parallel the snATAC-seq libraries were generated using the 10x Chromium Single Cell ATAC v1 with a target of 10,000 nuclei per library according to the manufacturer's instructions.