GSM9114312: Alkali burn-3, Day 14, rep3; Oryctolagus cuniculus; RNA-Seq
Rabbit corneas were harvested 14 days post-alkali injury (n=3) along with naïve controls (n=3) and processed into single-cell suspensions using an optimized enzymatic digestion protocol designed to preserve cellular integrity. Each eye was sectioned into eighths, and four pieces (half a cornea) were placed in Falcon™ Round-Bottom Polystyrene Tubes (Fisher Scientific, 14-959-5) containing 300 µL of digestion buffer at 4°C. The buffer comprised 2.5 mg Liberase TL (Thermo Fisher, 17101015), 2 mg DNase I (Fisher Scientific, AAJ62229MB), and 1 mL incomplete RPMI 1640 medium with L-glutamine (Thermo Fisher, 11875119). Tissues were finely chopped in the buffer and incubated at 37°C with shaking (400 rpm) for 30 minutes. Tissue clumps were disrupted using a 200 µL pipette tip and agitated every 5 minutes until full dissociation. To terminate enzymatic activity and stabilize cells, 300 µL of heat-inactivated fetal bovine serum (Thermo Fisher, 10082147) was added and mixed thoroughly. The suspension was pipetted repeatedly, filtered through a 70 µm Falcon™ Cell Strainer (Fisher Scientific, 08-771-2), and transferred to 15 mL tubes. Cells were washed three times with 3 mL of FACS buffer (PBS + 2% FBS), centrifuged at 200×g (1000 rpm) for 5 minutes with brake set to 1, and the supernatant was gently removed by pipetting. The final cell pellet was resuspended in 300 µL FACS buffer or adjusted based on concentration requirements. scRNA sequencing was conducted via the 10x Genomics Chromium platform following standard protocols for single-cell library preparation. The 10x Genomics single-cell 3' library preparation was performed by the Genomics Technology Core at the Bond Life Sciences Center. Sequencing was conducted on an Illumina NovaSeq X Plus instrument, generating 100 base-pair (bp), paired-end reads. The expected sequencing depth was ~50,000 read pairs per cell per library, optimized for 3' RNA sequencing, with a target of 10,000 single-cell transcriptomes per sample to ensure high coverage and resolution. The scRNA-Seq libraries were constructed via 10x Genomics Chromium Chip technology, with precise library preparation to ensure optimal cell recovery, minimal background noise, and accurate unique molecular identifier (UMI) processing.
GEO
GSM9114312_r1