GSM8956559: untreated anterior segment cells, rep2; Mus musculus; RNA-Seq
RNA-seq of sorted cells: eyes from Ai9 mice were cut circumferentially 1 mm posterior to the limbus to isolate the cornea, iris, and part of the ciliary body. Isolated tissues were digested with 625 units/mL of collagenase type I (Sigma-Alrich) in PBS for 45 minutes at 37°C followed by 0.25% Trypsin-EDTA (Gibco) for 10 minutes at 37°C to isolate single-cell suspensions, then washed with PBS and stained with 0.1 μg/mL of DAPI to exclude dead cells. tdTomato-positive cells were sorted from eyes that received Cre mRNA with CART 8. Two eyes were pooled for each sample and a minimum of 500 cells sorted. Anterior segment control cells were collected from eyes without treatment. One eye was used for each sample and 1,000 cells sorted. Total RNA from sorted cells was collected using a Quick-RNA Microprep Kit (Zymo). RNA-seq of whole anterior segments: eyes from C57BL/6J mice were cut circumferentially 1 mm posterior to the limbus to isolate the cornea, iris, and part of the ciliary body. Isolated tissues were homogenized using a handheld pellet pestle (Kimble Chase) in 300 μL of RNA Lysis Buffer (Zymo) and centrifuged at 16,000 x g for 5 minutes. Total RNA was collected from the supernatant using a Quick-RNA Microprep Kit (Zymo). Ultra-low input RNA-seq library construction and sequencing was performed by Azenta Life Sciences (South Plainfield, NJ, USA) with a target depth of 20 million reads per sample.