title: GSM9268159: B6_WT_2mo_ColumbiaU_scRNA_2; Mus musculus; RNA-Seq
library_construction_protocol: (Columbia University scRNA-seq samples): Tissues were digested enzymatically using Papain and Deoxyribonuclease I (Worthington Biochemical Corporation, Cat. no. LK003153) for 20 min at 37°C and stopped using Earl's balanced salt solution (EBSS, Thermo Fisher Scientific, Cat no. 24010-043). Cells were triturated using an 18-gauge needle, centrifuged at 300g at 4 °C, washed with cold DMEM, and filtered using a 100 μm Flowmi Cell Strainer (H13680-0040, Bel-Art. Cells were resuspended in cold DMEM and placed on ice immediately. (Columbia University multiome sample): Limbal strip samples for multiome analysis were snap-frozen on dry ice immediately following dissection and stored at -80C until processing. To isolate nuclei, tissue was homogenized in 100 μL of chilled lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween20, and 0.01% digitonin – from [142] supplemented with 1% BSA). Afterwards, samples were incubated for 5 min on ice, gently agitated by pipetting, and incubated for an additional 10 minutes on ice. We then added 1 mL of chilled wash buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20, 1% BSA) to the cells and centrifuged at 500 rcf for 5 min at 4°C. Cells were resuspended in chilled Diluted Nuclei Buffer (PN-2000153, 10x Genomics) and filtered through a 40 μm Flowmi Cell Strainer (H13680-0040, Bel-Art). Cells were resuspended in cold DMEM and placed on ice immediately. (Duke University scRNA-seq sample): Single cell dissociation was performed using Collagenase IV (Worthington Biochemical Cat. no. LS004188), Dispase II (Sigma Cat. No. D4693) and DNAse I (Roche Cat no. 50225500) for 60 min and then trypsin/EDTA for 10 min at 37°C. Cells were triturated using a 1 ml pipette tip, centrifuged at 300g at 4°C, washed with cold DMEM containing 10% FBS, and filtered using a 70 μm cell strainer (Fisher). Cells were resuspended in cold DMEM containing 5% FBS and placed on ice immediately Single-cell and single-nucleus RNA-seq was performed at the single-cell sequencing core in Columbia Genome Center (Columbia University) or Duke Molecular Genomics Core (MGC, Duke University). Single cells or nuclei were loaded into chromium microfluidic chips with v3 chemistry and barcoded with a 10x chromium controller (10x Genomics). RNA from the barcoded cells was reverse-transcribed, and sequencing libraries were constructed with a Chromium Single Cell v3 reagent kit (10x Genomics). Sequencing at both institutions was performed on NovaSeq 6000 (Illumina).