GSM5651516: 04-Epithelium; Homo sapiens; RNA-Seq
Manual dissection of the corneal tissue: Eight donor corneas were dissected for scRNAseq. First, cells were isolated from six corneas that were manually dissected to separate the epithelial, stromal and the endothelial layers to ensure the gentlest possible enzymatic dissociation of each layer. The corneas were vacuum fixed in a punch base (e.janach) endothelial-cell side up, stained with trypan blue solution (0.4%) for 30 s and washed with BSS ophthalmic irrigation solution. The corneal endothelium was then gently lifted following the Schwalbe line using a DMEK cleavage hook (e.janach) and fully stripped using angled McPherson tying forceps. The remaining tissue was trephined using a 9.5 mm Ø Barron vacuum punch (Katena) in order to separate the epithelium and stroma from the scleral ring. The remaining two corneas were used for limbus isolation. A surgical scalpel was used to cut the limbus into approximately 1 × 2 mm fragments, which were then rinsed with PBS.    Tissue dissociation to single cells: The six manually dissected corneal tissues were enzymatically treated to obtain single cell suspensions. The stripped corneal endothelium was incubated with 2 mg/mL collagenase (Sigma) solution in human endothelial serum free media (SFM) (Thermo Fisher Scientific) for 1–2 h at 37°C followed by a 10 min incubation with Accutase (Thermo Fisher Scientific) at 37°C to obtain a single cell suspension. The cells were centrifuged for 5 min at 800 × g and resuspended in 0.5 mL of human endothelial SFM media.    The corneal epithelium–stroma tissues were treated with 1.5 mg/mL collagenase and 0.2 mg/mL bovine testis hyaluronidase (Sigma) in DMEM/F12 (Thermo Fisher Scientific) for approximately 1 h at 37 °C. The released corneal epithelium was suctioned with a P1000 micropipette and further treated with Accutase for 10 min at 37°C to obtain a single cell suspension of corneal epithelial cells. The cells were centrifuged for 5 min at 800 × g and resuspended in 0.5 mL of DMEM/F12. The remaining corneal stroma was treated with 1.5 mg/mL collagenase and 0.2 mg/mL bovine testis hyaluronidase in DMEM/F12 for 5–7 h to obtain a single cell suspension of corneal keratocytes. After the incubation, the cells were centrifuged for 5 min at 800 × g and resuspended in 0.5 mL of DMEM/F12 media.    The limbus fragments isolated from the two corneas for limbal isolation were digested with four trypsinization cycles. In each cycle, the limbus fragments were incubated in 10 mL of 0.05% trypsin/0.01% EDTA (Thermo Fisher Scientific) at 37°C for 30 min. The trypsin containing dissociated cells were transferred to a 50 mL centrifuge tube containing 10 mL DMEM/F12 media and centrifuged for 5 min at 300 × g, after which the cells were resuspended in 0.5 mL of DMEM/F12 media. The undigested limbal fragments were placed again in 10 mL of 0.05% trypsin/0.01% EDTA for the following trypsinization cycle. The cells isolated from the limbus of the two donor corneas were pooled for the following steps.    Methanol cell fixation  After dissociation, the single cell suspensions were passed through a 100 μm Ø cell strainer, centrifuged for 5 min at 300 × g and resuspended in 1 mL ice-cold PBS to eliminate any medium remnants. Next, the cells were again centrifuged for 5 min at 300 × g and resuspended in ice-cold PBS at a ratio of 200 μL PBS per 1×10^6 cells followed by the dropwise addition of ice-cold methanol, at a ratio of 800 μL per 1×10^6 cells. The cells were stored at -80°C until sequencing. Single-cell mRNA sequencing was performed at Single Cell Discoveries according to standard 10x Genomics 3' V3.1 chemistry protocol. Prior to loading the cells on the 10x Chromium controller, cells were rehydrated in rehydration buffer. Cells were then counted to assess cell integrity and concentration. Approximately 9,000 cells (comprising 3,000 cells from each of the three layers) from corneas 01, 02, 03 and 04 were separately loaded by layer and cornea. Furthermore, 3,000 cells (1,000 cells from each of the three layers) from corneas 05 and 06, and 3,000 cells from corneas 07 and 08 (from the limbal samples) were separately loaded by cornea. The resulting sequencing libraries were prepared following a standard 10x Genomics protocol.