GSM8509015: Retina,2.5mo,donor 2,snATAC; Mus musculus; ATAC-seq
The flash frozen retinal tissue along with RPE and choroid was harvested from 28days, 2.5month, 5month and 21month old mice. Each age point contained two biological replicates. Nuclei preparation was adapted from Lacar et al. 2016 (PMID: 27090946). Flash frozen mouse retina was resuspended in 1 mL of  douncing buffer consisting of 0.25M sucrose (S1888, Sigma), 25mM KCl (AM9610G, Invitrogen), 5mM MgCl2 (194698, Mp Biomedicals Inc.), 10 mM Tris-HCl pH 7.5 (15567027, Thermo Fisher Scientific), 1mM DTT (D9779, Sigma), 1X protease inhibitor (05056489001, Roche), 0.1% Triton X-100 (T8787-100ML, Sigma), and 0.5 U/ul RNasin RNase inhibitor (PAN21110, Promega) in molecular biology grade water (46000-CM, Corning). Frozen tissue was then transferred to a dounce homogenizer on ice and dounced 25 times with a loose plunger and 25 times with a tight plunger. Suspension was then passed through a Celltrix 30 μM filter (04-004-2326, Sysmex) and washed with 300 mL of douncing buffer before being centrifuged for 10 minutes at 1000 rcf in a swinging bucket centrifuge at 4°C with run setting 3/3. Pellet was then washed with an additional 1 mL of douncing buffer without Triton X-100, and split for single-nucleus ATAC-seq and single-nucleus RNA-seq in a ratio of 700 mL /300 mL. Nuclei from dissociated tissue were permeabilized in 1 mL of  permeabilization buffer (5% BSA, 0.2% IGEPAL-CA630, 1 mM DTT, 1x cOmplete EDTA-free protease inhibitor in PBS), pipette mixed, and incubated on ice for 10 minutes. Nuclei were then centrifuged at 500 rcf for 5 minutes and the supernatant was removed. Nuclei were resuspended in 500 µL high salt tagmentation buffer (36.3 mM Tris-acetate (pH = 7.8), 72.6 mM potassium-acetate, 11 mM Mg-acetate, 17.6% DMF) and counted using a hemocytometer. Concentration was adjusted to 2000 nuclei/9 µL, and 2000 nuclei were dispensed into each well of one 96-well plate. For tagmentation, 1 µL barcoded Tn5 transposomes (58) was added using a BenchSmart 96 (Mettler Toledo), mixed five times and incubated for 60 min at 37°C with shaking (500 rpm). To inhibit the Tn5 reaction, 10 µL of 40 mM EDTA were added to each well with a BenchSmart 96 (Mettler Toledo) and the plate was incubated at 37°C for 15 min with shaking (500 rpm). Next, 10 µL 3x sort buffer (2% BSA, 2 mM EDTA in PBS) was added using a BenchSmart 96 (Mettler Toledo). All wells were combined into a FACS tube and stained with 3 µM Draq7 (Cell Signaling). Using a SH800 (Sony), 20 2n nuclei were sorted per well into eight 96-well plates (total of 768 wells) containing 10.5 µL EB (25 pmol) primer i7, 25 pmol primer i5, 200 ng BSA (Sigma). Preparation of sort plates and all downstream pipetting steps were performed on a Biomek i7 Automated Workstation (Beckman Coulter). After addition of 1 µL 0.2% SDS, samples were incubated at 55°C for 7 min with shaking (500 rpm). 1 µL 12.5% Triton-X was added to each well to quench the SDS. Next, 12.5 µL NEBNext High-Fidelity 2 × PCR Master Mix (NEB) were added and samples were PCR-amplified (72°C 5 min, 98°C 30 s, (98°C 10 s, 63°C 30 s, 72°C 60 s)×12 cycles, held at 12°C). After PCR, all wells were combined. Libraries were purified according to the MinElute PCR Purification Kit manual (Qiagen) using a vacuum manifold (QIAvac 24 plus, Qiagen) and size selection was performed with SPRI Beads (Beckmann Coulter, 0.55x and 1.5x). Libraries were purified one more time with SPRI Beads (Beckmann Coulter, 1.5x).