GSM8678203: P0, chicken, snATAC-seq; Gallus gallus; ATAC-seq
Chicks were euthanized by carbon dioxide inhalation followed by manual cervical dislocation. Chicken eyes were harvested and chicken retinas were removed from the eye by dissection, transferred to calcium and magnesium-free Hank's balanced salt solution (Thermo Fisher Scientific) and dissociated into single cells by papain digestion. A single chick retina was incubated in 800 μl of HBSS with 1.3 mg of papain (Worthington Biochemical Corporation) at 37°C for 10 min. The retina was then dissociated by gentle trituration with a pipette. The dissociated cells were washed with Dulbecco's Modified Eagle Media (DMEM; Thermo Fisher Scientific) containing 10% fetal bovine serum (Thermo Fisher Scientific) and DNaseI (Roche) for 5 min at 37°C. The cells were then pelleted by centrifugation at 1000xg for one minute. The cells were then resuspended in Dulbecco's phosphate-buffered saline (DPBS) with 1% BSA and filtered for nuclei isolation. For nuclei isolation, the dissociated retinal cells were subjected to centrifugation at 300xg for 5 minutes at 4°C. The cell pellet was then resuspended in 100 µl of lysis buffer (0.1% IGEPAL CA-630, 0.1% Tween-20, and 0.01% digitonin in lysis dilution buffer [10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 1% BSA, pH 7.4]), mixed three times by pipetting, and incubated on ice for 3 min. Subsequently, the nuclei were added to 1 ml of wash buffer (0.1% Tween-20 in lysis dilution buffer) and centrifuged at 500xg for 5 min at 4°C. Finally, the nuclei pellet was resuspended in 50 µL of 1x Diluted Nuclei Buffer (10x Genomics) and filtered through a 40 µm Flowmi™ Cell Strainer. The nuclei were enumerated on a hemocytometer. The cell nuclei (~22,000 input nuclei) were then resuspended and used for transposition, subsequently loaded into the 10x Genomics Chromium Single Cell system. One scATAC-seq library was constructed using the Chromium Single Cell ATAC Reagent Kits v1.1 (10x Genomics), following the manufacturer's instructions.