GSM7431852: AI_2; Taeniopygia guttata; RNA-Seq
Frontal slices (400 μm thick, one slice per hemisphere) containing RA, AId, and AIv were cut on a Leica VT1200S vibratome in an ice-cold cutting solution containing 119 mM NaCl, 2.5 mM KCl, 8 mM MgSO4, 16.2 mM NaHCO3, 10 mM HEPES, 1 mM NaH2PO4, 0.5 mM CaCl2, 11 mM D-Glucose, 35 mM Sucrose with a pH of 7.3-7.4 when bubbled with carbogen (95% O2, 5% CO2; ~330-340 mOsm). Slices were then transferred to an incubation chamber containing artificial cerebral spinal fluid with 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 26.2 mM NaHCO3, 1 mM NaH2PO4, 1.5 mM CaCl2, 11 mM D-Glucose, 35 mM Sucrose with a pH of 7.3-7.4 when bubbled with carbogen (95% O2, 5% CO2; ~330-340 mOsm) at 37°C. Microbiopsies containing RA, AId, and AIv were dissected using a scalpel and angled forceps and flash frozen in a dry ice/isopropyl alcohol slurry. Tissue was homogenized in 300 µL of nuclei lysis buffer (10 mM Tris pH 8.0, 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 0.1% Triton-X 100, 0.5% RNasin, and 0.1 mM DTT). 700 µL of lysis buffer was added to the homogenate and the sample was incubated on ice for 5 min. The sample was transferred to a 15 ml conical tube and centrifuged at 500 x g for 5 min at 4°C. The supernatant was discarded, 1 ml of ice-cold lysis buffer was added, and the mix was incubated on ice for 5 min. The sample then was centrifuged at 500 x g for 5 min at 4°C. The pellet was resuspended in 1 ml of nuclei suspension buffer (1x PBS, 0.01% BSA, and 0.1% RNasin), and filtered through a FLOWMI 40 μm tip strainer (Bel-Art) and centrifuged at 500 x g for 5 min at 4°C. The pelleted nuclei were resuspended in nuclei suspension buffer. Libraries were prepared using the Chromium Single Cell 3' Library & Gel Bead Kit v3 (10x Genomics) according to the manufacturer's instructions