GSM9872460: Multiome, retina, GEX; Mus musculus; RNA-Seq
Eight retinas from 11- and 93-week-old C57BL/6 mice (4 per age group) were rapidly dissected from enucleated eyes in cold PBS, immediately snap-frozen on dry ice, and stored at -80°C. Snap-frozen retinal tissue was transferred into a glass Dounce homogenizer containing 500 µL chilled 0.1× lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.01% Tween-20, 0.01% Nonidet P40 Substitute, 0.001% digitonin, 1% BSA, 1 mM DTT, 1× RNase inhibitor in nuclease-free water) and immediately homogenized. Homogenates were incubated on ice for 5 minutes, gently triturated using a wide-bore pipette tip, and incubated on ice for an additional 10 minutes. Following lysis, 500 µL of chilled wash buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 1% BSA, 1 mM DTT, 1× RNase inhibitor in nuclease-free water) was added, and the samples were mixed gently. Nuclei suspensions were sequentially filtered through 70 µm and 40 µm Flowmi cell strainers to remove debris and aggregates, centrifuged at 500 × g for 5 minutes at 4°C, washed twice with chilled wash buffer, and resuspended in chilled diluted nuclei buffer (1× Nuclei Buffer with 1 mM DTT and 1× RNase inhibitor). Single-nucleus ATAC and gene expression libraries were constructed jointly using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics) according to the manufacturer's protocols. Ten thousand nuclei were targeted for library preparation. Nuclei suspensions were loaded using the Chromium Next GEM Chip J Single Cell Kit (10X Genomics). The resulting libraries were sequenced on an Illumina NovaSeq 6000 platform targeting a depth of 25,000 read pairs per nucleus.