title: GSM9034552: Donor2046_mRNA; Homo sapiens; OTHER
library_construction_protocol: Human corneal–limbal and conjunctival tissues were incubated separately at 37 °C for 2 hours in DMEM/F12 containing 2.4 IU/mL Dispase II (Thermo Fisher Scientific, Waltham, MA, USA). The epithelia were scraped, collected, and further digested with Gibco™ TrypLE (Thermo Fisher Scientific) for 5 minutes. The remaining stromal and conjunctival fragments were cut into small pieces, placed each in a MACS C tube (Miltenyi Biotec, Bergisch Gladbach, Germany) containing 4 mg/mL collagenase A (Sigma-Aldrich, St. Louis, MO, USA) in DMEM, and digested for 1 hour at 37 °C using a gentleMACS dissociator (Miltenyi Biotec). After digestion, single cells from all individual compartments were pooled, filtered through a 70 µm cell strainer (Miltenyi Biotec), washed in PBS supplemented with 0.04% bovine serum albumin (BSA; Sigma-Aldrich), and counted. Cells were resuspended in cold 0.04% BSA-PBS, and cold methanol was added dropwise to a final concentration of 80%. Samples were incubated at –20 °C for 30 minutes before storage at –80 °C.  Mouse conjunctival and corneal tissues were digested separately in DMEM containing 2 mg/mL collagenase A and 10 mg/mL Dispase II at 37 °C for 1 hour. During digestion, tissues were gently triturated by passing them through a 20G syringe needle five times every 20 minutes. Following enzymatic digestion, samples were rinsed with DMEM and incubated in TrypLE (Thermo Fisher Scientific) for 10 minutes at 37 °C. Cell suspensions were filtered through a 70 µm cell strainer, washed in 0.04% BSA-PBS, and counted. Cells were resuspended in cold 0.04% BSA–PBS, and cold methanol was added dropwise to a final concentration of 80%. Samples were incubated at –20 °C for 30 minutes before storage at –80 °C. Single-cell suspensions (1 mL, approx. 106 cells/mL) were thawed and washed in wash-resuspension buffer containing 3× SSC, 1 mM DTT, 0.04% ultrapure BSA, and 0.2 U/μL RNase inhibitor. Cells were incubated on ice for 30 minutes with a mixture of DNA-labeled lectins and biotinylated SNA I, each at 0.5 μg/mL (equivalent to 0.05 μg of lectin per million cells). After three washes in wash-resuspension buffer, cells were incubated with TotalSeq™-B0975 Streptavidin (BioLegend, San Diego, CA) at 0.5 μg/mL (0.05 μg per million cells) for 30 minutes on ice. Cells were then washed three times, counted, and adjusted to a final concentration of ~1,000 cells/μL. Single cells were captured using the Chromium Controller, and single-cell RNA sequencing libraries were prepared with the Chromium Next GEM Single Cell 3′ v3.1 Kit and the 3′ Feature Barcode Kit (10x Genomics, Pleasanton, CA, USA) according to the manufacturer's instructions to generate both the gene expression and cell surface feature libraries. For sequencing, gene expression and cell surface feature libraries from each sample were pooled at a 4:1 ratio and sequenced using either the Illumina NovaSeq 6000 or NovaSeq X Plus platforms, targeting 20,000 reads per cell.