GSM9455531: Multiome, Bengalese finch NC, GEX, biol rep 2; Lonchura striata; RNA-Seq
For snRNA/ATAC-seq samples, three to four adult (>120 dph) male Bengalese finches were anesthetized with isoflurane, transcardially perfused with ice-cold carbogenated ACSF, then decapitated. Brains were dissected out into ice-cold carbogenated ACSF then coronally sectioned 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. Nuclei were isolated using a modified version of the Optiprep method. Several stable buffers were prepared before nuclei isolation and stored a 4C: 1.079x Homogenization Buffer Stable Solution ("HBS", 260 mM sucrose, 30 mM KCl, 10 mM MgCl2, 20 mM Tricine-KOH pH 7.8), Diluent Buffer (150 mM KCl, 30 mM MgCl2, 120 mM Tricine-KOH pH 7.8), 50% iodixanol solution (diluted from 60% Optiprep Density Gradient Medium, Sigma, D1556, using Diluent Buffer), and 5X ATAC-RSB (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 15 mM MgCl2). On the day of nuclei isolation, the following buffers were prepared: 1x Homogenization Buffer Unstable Solution ("HBU", HBS to 1x, 1 mM DTT, 0.5 mM spermidine, 0.15 mM spermine, 0.3\% NP-40, 1 U/uL Protector RNase Inhibitor (Sigma 3335402001), 1x Complete Protease Inhibitor (prepared from a 25x solution consisting of one tablet (Sigma, 11697498001) dissolved in 2 mL nuclease-free water)), 30% Iodixanol Solution and 40% Iodixanol Solution (both diluted from 50% iodixonal solution using 1x Homogenization Buffer Unstable Solution), and ATAC-RSB-Tween (1x ATAC-RSB, 0.1\% Tween-20, 1% BSA, and 1 U/uL Protector RNase Inhibitor). Samples from different birds were pooled and 1 mL 1x HBU was added. Samples were incubated on ice for 5 minutes, then transferred to a 2 mL glass dounce (Kimble). Samples were homogenized with 15 strokes of the loose pestle, then 15 strokes of the tight pestle. Samples were transferred to 2 mL tubes then centrifuged for 5 minutes at 350xg at 4C using a swing-out rotor. All but 200 uL of supernatant was removed, the total volume was brought up to 400 uL using 1x HBU, then the pellet was gently resuspended by pipetting. 400 uL 50% iodixanol solution was added and mixed well by pipetting. 600 uL 30% iodixanol solution was layered underneath the 25% iodixonal/tissue layer, followed by 600 uL 40% iodixanol solution underneath the 30% layer. In a pre-chilled swing-out rotor, samples were centrifuged for 20 minutes at 10,000xg at 4C with the brake off. Starting at the top of the solution, enough solution was aspirated off to leave ~200-300 uL above the interface between the 30% and 40% iodixanol layers, which contains nuclei. 200 uL of the nuclei band was aspirated and transferred to a clean 1.5 mL tube containing 1 mL of ATAC-RSB-Tween. Nuclei were centrifuged for 10 minutes at 500xg at 4C using a swing-out rotor. The entire supernatant was carefully removed, followed by addition of 12 uL Nuclei Resuspension Buffer (10X Genomics) and gentle resuspension by pipetting. Nuclei were counted and their concentrations were adjusted to 800-12,000 nuclei/uL. The nuclei were then loaded onto a Next GEM Chip J (10X Genomics) at numbers targeting 2,500 to 10,000 recovered nuclei. Sequencing libraries for each region were prepared using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit. The libraries were then pooled, and sequenced on an Illlumina NovaSeq S4 200 flow cell at the UCSF CAT.