GSM4649016: HVC_1; Taeniopygia guttata; RNA-Seq
Birds were rapidly decapitated and brains were dissected out and placed in ice-cold ACSF (126 mM NaCl; 3 mM KCl; 1.25 mM NaH2PO4; NaHCO3; 10 mM D-(+)- glucose, 2 mM MgSO4; 2 mM CaCl2) bubbled with carbogen gas (95% O2, 5% CO2). Each hemisphere was glued to a specimen tube and sectioned with a VF-200 Compresstome (Precisionary Instruments). Sagittal 200 µM slices were made in ice-cold ACSF. Brain regions were dissected by hand with scissors under a dissecting microscope and then placed in a tube with ACSF over ice. Each tube contained 1-3 samples of a brain region from the same hemisphere of a single individual. When all tissue was collected, excess ACSF was removed from each tube, samples were flash-frozen in liquid nitrogen, and stored at -80 °C. For nuclei extraction, samples were thawed over ice, brain regions were then pooled and dounce-homogenized in 500 µL of ice-cold Lysis Buffer (10 mM Tris-HCL; 10 mM NaCL; 3 mM MgCl2; 0.1% IGEPAL CA-630), samples were transferred to clean 1.5 mL tubes. 900 µL of 1.8 Sucrose Cushion Solution (NUC201-1KT, Sigma, MO, USA) was added to each tube and pipette mixed ten times. In a clean 2 mL tube, 1400 µL of nuclei sample was layered on top of 500 µL Sucrose Cushion Solution without mixing. Samples were centrifuged at 13,000 x g for 45 min at 4 °C. 300 µL Nuclei Suspension Buffer (1% UltraPure BSA (AM2618, Thermo Fisher Scientific, MA, USA) and 0.2 RNase inhibitors (Clontech, #2313A) in PBS) was added to 100 µL supernatant and re-suspended. Samples were then centrifuged at 550 x g for 5 min at 4 °C. All but 50 µL supernatant was discarded and the nuclear pellet was resuspended and filtered through a FLOWMI tip strainer (Bel-Art Products, #H13680-0040, NJ, USA) into a new tube. Nuclei were counted in a 1:20 dilution of nuclei solution in Trypan Blue loaded onto a hemocytometer. Libraries were prepared using the Chromium Single Cell 3' Library & Gel Bead Kit v3 according to the manufacturer's instructions and sequenced using an Illumina NovaSeq 6000 at the North Texas Genome Center at UT Arlington with 28 cycles R1 and 124 cycles R2. Followed 10X Genomics kit manufacturer guidelines.
GEO Accession
GSM4649016