title: GSM6923655: Human_1; Homo sapiens; RNA-Seq
library_construction_protocol: Corneal endothelium samples were extracted from the tissue and sequenced as follows:  Isolation of human corneal endothelium  Of the five human corneal endothelial samples, four were discarded peripheral endothelial rims (1.5 mm wide) resulting from tissue preparation for Descemet's membrane endothelial Keratoplasty (DMEK) according to clinical practice (Català et al. 2020) and one (Human 5) was a whole endothelium stripped from a cornea deemed unsuitable for transplant (Table 1).     The four endothelial rims were lysed in 300 μL trizol within 48 h after tissue preparation. From the whole cornea, the complete endothelium was manually stripped following lysis in 300 μL trizol. Briefly, the cornea was vacuum-fixed in a punch base (e.janach) endothelial-cell side up and trephined with a 10 mm Ø corneal punch at a fixed depth of 100 μm (e.janach). To delimit the endothelial trephined line, the cornea was stained with a trypan blue solution (0.4%) for 30 s, and washed with balanced salt solution (BSS) ophthalmic irrigation solution. The corneal endothelium was then gently lifted using a DMEK cleavage hook (e.janach), fully stripped using angled McPherson tying forceps, and immediately transferred to trizol. All sample lysates were stored at -80 °C until further use.    Isolation of sheep corneal endothelium  All samples (n=5) were obtained from Texel sheep used for a dorsal titanium implant study from the animal facility at Maastricht University (Table 1). Five eyes were enucleated from 1 to 2-year-old female Texel sheep within 4 h of sacrifice, and corneoscleral disks were excised from whole ocular gloves. Corneal endothelial cells were enzymatically treated and scrapped from the corneoslceral disks because stripping of the endothelium proved not successful due to the tissue characteristics. For this purpose, corneoscleral disks were rinsed with Dulbecco's phosphate buffered saline (DPBS), vaccum-fixed in a punch base (e.janach) endothelial-side up and treated with approximately 150 μL of StemPro Accutase (Thermo Fisher Scientific) for 20 min at ambient temperature. Corneal endothelial cells were then gently scraped using a DMEK cleavage hook (e.janach) and rinsed off the corneoscleral disk with approximately 2 mL of MEM (Thermo Fisher Scientific). The cell suspension was then transferred to a 15 mL tube, centrifuged at 800 × g for 5 min and lysed in 300 μL trizol. All sample lysates were stored at -80 °C until further use.    Isolation of rabbit corneal endothelium  Eyes were obtained from New Zealand White rabbits used for other experimental purposes from the Utrecht University animal facility (n=3) and MSD Animal testing facility (n=2) in Boxmeer, the Netherlands (Table 1). Eyes were enucleated 30 min to 4 h after sacrifice, corneoscleral disks were excised from whole ocular globes, and corneal endothelium stripped. In brief, corneoscleral disks were rinsed with PBS, vacuum-fixed in a punch base endothelial-side up and trephined with a 10 mm Ø corneal punch at a fixed depth of 100 μm. To delimit the endothelial trephined line, the cornea was stained with a trypan blue solution (0.4%) for 30 s, and washed with BSS ophthalmic irrigation solution. The corneal endothelium was then gently lifted using a DMEK cleavage hook, fully stripped using angled McPherson tying forceps, and immediately transferred to 300 μL trizol. All sample lysates were stored at -80 °C until further use. RNA isolation and bulk RNA sequencing  RNA isolation was performed following trizol isolation procedure, and RNA integrity was assessed with a 2100 Bioanalyzer (Agilent). RNA sequencing was performed at Single Cell Discoveries (Utrecht, the Netherlands). Libraries were prepared following CEL-seq2 protocol (Hashimshony et al. 2016) to enable sample multiplexing. Paired-end sequencing was performed on Illumina Nextseq500, high output 2 × 75 bp run mode, at a sequencing depth of 20 million reads/sample.     Data analysis and pre-processing  BCL files resulting from sequencing were transformed to FASTQ and read 1 was used to identify the Illumina library index and CEL-seq sample barcode. Read 2 was aligned to the specific RefSeq reference genome of each species, namely: hg19 for human, OryCun2.0 for rabbit, and ARS-UI_Ramb_v2.0 for sheep using STAR genome aligner (Dobin et al. 2013).   The data from all samples were loaded in R (version 4.1.2) (R Core Team 2013) to perform analysis. Gene homology mapping from rabbit and sheep to human was performed using the orthogene R-package (version 1.1.0) with the g:profiler method (Raudvere et al. 2019). The three datasets were combined and only genes with homologs in all three species were included in the downstream analyses. Once the data was combined quantile normalization was performed using the limma package (version 3.52.2) (Ritchie et al. 2015).