GSM9455541: snRNA-seq, RA; Lonchura striata; RNA-Seq
Adult (>120 days post hatch) male Bengalese finches were euthanized via isoflurane overdose then decapitated. The brain was dissected out into ice-cold ACSF (152 mM choline chloride, 2.5 mM KCl, 1.1 mM NaH2PO4, 25 mM NaCO3, 35 mM glucose, 10 mM HEPES, 3 mM MgSO4*7H2O, and 0.5 mM CaCl2*2H2O) bubbled with carbogen. The brain was then sectioned in ice-cold carbogenated ACSF either coronally or sagittally using a Leica VT1000 vibratome at 300 um. Areas of interest were microdissected out, placed in cryovials, frozen in liquid nitrogen, then transferred to -80C for long-term storage.  500 uL ice-cold lysis buffer (10 mM Trizma pH 7.4 (Sigma, T2194), 10 mM NaCl, 3 mM MgCl2, and 0.1% NP-40) was added to each cryovial, and the tissue was transferred to 2 mL glass dounces (Kimble). Samples were dounced 15 times using a loose pestle then 15 times using a tight pestle. 900 uL sucrose cushion buffer (Sigma, NUC201-1KT) was added to each sample and was slowly mixed by pipetting 20 times. 500 uL sucrose cushion buffer was added to a new 2 mL tube, then the 1400 uL of tissue/lysis/sucrose bufffer was carefully layered on top. Samples were centrifuged for 45 minutes at 13,000xg at 4C. All but 100 uL supernatant was discarded, 300 uL nuclei suspension buffer (1x PBS, 1% UltraPure BSA (ThermoFisher, AM2616), and 0.5% RNase inhibitor (Clontech, 2313A)) was added, then the pellet was gently resuspended by pipetting. Samples were centrifuged for 5 minutes at 550xg at 4C. All but 70 uL of supernatant was discarded, the pellet was resuspended, and the sample was passed through a 40 um FlowMi filter (Bel-Air). Nuclei were counted on a C-Chip and standardized to 400-700 nuclei / uL. Nuclei were added to target recovery 10,000 nuclei onto a Next GEM Chip G microfluidics chip as part of the Next GEM Single Cell 3ʹ Kit v3.1 (10X Genomics). Following multiplexing, libraries were sequenced on two lanes of an Illumina NovaSeq S4 200 flow cell (101 cycles R1, 12 cycles I1, 24 cycles I2, 101 cycles R2) at the UCSF Center for Advanced Technology.