GSM8519435: RPE, donor 1, multiome RNA; Homo sapiens; OTHER
The eye globes were collected from three unrelated human donors (aged 21, 27 and 43 years) with the collaboration of San Diego Eye Bank. The sample processing time starting from eye enucleation to dissection was restricted within 2 hours to identify the macula, a visible yellowish/whiteish indent that is slightly lateral to the optic disk inside the globe. Prior dissection, the eye globe with proper label was collected in a sterile moist chamber. At first the cornea was removed by cutting 2-3 mm around the limbus using a surgical blade. Pull off the iris and lens. This will leave you with the eye cup, which contains the posterior pole. After removing enough vitreous and aqueous fluid the temporal region can be located by two main blood vessels that curl around the macula. Using a sterile 3mm skin biopsy punch, the macula was collected. Sections were made for the other regions, temporal, nasal, superior and inferior. For all these regions, the retina and RPE tissue were separated and stored immediately in Liquid Nitrogen. Retina tissues were pulverized using a dounce tissue chamber and two pestles (“Loose” and “Tight”) on ice for each sample. Pulverized retina tissues were resuspended in 1ml of chilled NIM-DP-L buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl pH 7.5, 1 mM DTT, 1x protease inhibitor (Pierce), 1U μl-1 recombinant RNase inhibitor (Promega, PAN2515) and 0.1% Triton X-100). Tissue was dounce homogenized with a loose pestle (5-10 strokes) followed by a tight pestle (15-25 strokes) or until the solution was uniform. Nuclei were filtered using a 30 μm CellTrics filter (Sysmex, 04-0042-2316) into a LoBind tube (Eppendorf, 22431021). Nuclei were centrifuged 1000 rcf, for 10 min at 4°C then the pellet was resuspended in 1ml NIM-DP buffer (0.25 M sucrose, 25 mM KCl, 5mM MgCl2, 10 mM Tris-HCl pH 7.5, 1mM DTT, 1x protease inhibitor, 1U μl-1 recombinant RNase inhibitor) and pelleted (1000 rcf, 10 min at 4°C). Pelleted nuclei were resuspended in 400 μl 2 μM 7-AAD (Invitrogen, A1310) in sort buffer (1mM EDTA, 1U μl-1 recombinant RNase inhibitor, 1x protease inhibitor, 1% fatty acid-free BSA in PBS). A total of 150,000 nuclei was sorted (Sony, SH800S) into a LoBind tube containing a collection buffer (5U μl-1 recombinant RNase inhibitor, 1x protease inhibitor, 5% fatty acid-free BSA in PBS). Then, 5x permeabilization buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 15 mM MgCl2, 0.05% Tween-20, 0.05% IGEPAL, 0.005% Digitonin, 5% fatty acid-free BSA in PBS, 5mM DTT, 1U μl-1 recombinant RNase inhibitor, 5x protease inhibitor) was added for a final concentration of 1x. Nuclei were incubated in ice for 1 min, then centrifuged (500 rcf, 5 min at 4°C). The supernatant was discarded and 650 μl of wash buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% fatty acid-free BSA in PBS, 1mM DTT, 1U μl-1 recombinant RNase inhibitor, 1x protease inhibitor) was added without disturbing the pellet followed by centrifuging(500 rcf, 5 min at 4°C). The supernatant was removed, and the pellet was resuspended in 7μl of 1x nucleus buffer (nucleus buffer (10x Genomics), 1mM DTT, 1U μl-1 recombinant RNase inhibitor). Nuclei (1μl) were diluted in 1x nucleus buffer, stained with Trypan Blue (Invitrogen, T10282) and counted, In total, 16000-20000 nuclei were used for the tagmentation reaction and controller loading and libraries were generated according to the manufacturer's recommended protocol (https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression).
GEO
GSM8519435_r1