title: GSM9674803: snRNA-seq, MAFB uninduced; Gallus gallus; RNA-Seq
library_construction_protocol: For snRNA-seq, microdissected tissue from chicken embryos was pooled by condition (e.g. tissue from all pPIDNB2-MAFB electroporated, doxycycline treated individuals was combined) before proceeding to nucleus isolation. 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, 0.2 U/uL Protector RNase Inhibitor (Sigma 3335402001, 10 mg/mL Kollidon VA-64 (Sigma, 190845)), 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 4degreeC 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. Following isolation, nuclei were washed in 1 mL of Illumina Nuclei Suspension Buffer (NSB) and pelleted by centrifugation at 500 × g for 5 minutes at 4℃ according to Illumina Single Cell 3′ RNA Prep protocol. Nuclei were resuspended in 20uL of NSB before counting. Nuclei were counted Revvity Cellometer K2 Fluorescent Cell Counter and diluted to 2500 nuclei/uL in NSB for samples at higher concentrations. snRNA-seq libraries were prepared according to the Illumina Single Cell 3′ RNA Prep, T2 protocol. Reactions were overloaded with 10,000 nuclei for samples whose concentrations supported this. Library concentrations were quantified using NEBNext Library Quant Kit for Illumina (NEB, E7630) before pooling. Libraries were sequenced on an AVITI PE150 flow cell at the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center.