title: GSM4040723: Area X_knockdown; Taeniopygia guttata; RNA-Seq
library_construction_protocol: Each bird was rapidly decapitated and its brain was placed in ice-cold ACSF (126 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM D-(+)-glucose, 2 mM MgSO4, 2 mM CaCl2) bubbled with carbogen gas (95% O2, 5% CO2). The cerebellum was removed with a razor and the cerebrum was glued to a specimen tube for sectioning with a VF-200 Compresstome (Precisionary Instruments). Coronal 500 µm sections were made in ice-cold ACSF and allowed to recover in room temperature ACSF for 5 min. Area X punches were collected into a tube containing ACSF on ice until all punches were collected and pooled from 2 birds per condition. Tissue punches were dounce-homogenized in 500 µl ice-cold Lysis Buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) and transferred to a clean 2 ml tube. Then 900 µl of 1.8 M Sucrose Cushion Solution (#NUC201-1KT, Sigma) was added and pipette-mixed with nuclei 10 times. To a second clean 2 ml tube, 500 µl of 1.8 M Sucrose Cushion Solution was added, and the nuclei sample was layered on top of the cushion without mixing. The sample was centrifuged at 13,000 × g for 45 min at 4°C and all but ~100 µl of supernatant was discarded to preserve the pellet. The pellet was washed in 300 µl Nuclei Suspension Buffer (NSB) (1% UltraPure BSA (#AM2618, Thermo Fisher Scientific) and 0.2% RNase inhibitors in PBS) and centrifuged at 550 × g for 5 min at 4°C. All but ~50 µl of supernatant was discarded and the pellet was resuspended in the remaining liquid and filtered through a FLOWMI 40 µm tip strainer(#H13680-0040, Bel-Art). Samples were diluted to 1000 nuclei/µl with NSB for targeting 10,000 nuclei for snRNA-seq. 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. Followed 10X Genomics kit manufacturer guidelines.