GSM5133875: WT mouse iridocorneal angle and limbal region wt185; Mus musculus; RNA-Seq
To avoid melanin aggregation during tissue dissociation and sample preparation, Angpt1ΔNC mice were crossed for 2 generations onto WT ICR mice to obtain litters of albino control and mutant animals. Animals were then aged to 6 weeks and groups of 2-3 Angpt1ΔNC and control mice were euthanized for pooled tissue collection. Male and female animals were pooled for analysis, resulting in two pooled control samples and a single pooled Angpt1ΔNC sample. 4-6 enucleated eyes were immediately dissected in ice-cold HBSS (Gibco-Life Technologies, Carlsbad CA) to obtain the isolated limbal-iridocorneal angle region. First, eyes were bisected on the saggital plane before the lens and retina were removed. The sclera was then removed by cutting immediately posterior to the ciliary body. Finally, the cornea was separated by cutting just anterior to the limbus, avoiding the TM. The majority of the iris was then removed, taking care to leave the ciliary body and TM intact. Limbal regions were then transferred to ice-cold DMEM containing 10% FBS and minced using fine scissors. After mincing, 1mg/ml Collagenase A (Worthington, Lakewood NJ) and 10μM Y27362 were added and tissues were transferred to a tissue culture incubator for 2 hours at 37°/5% CO2. After incubation, tissues were washed 1x in PBS before incubation in 0.25% Trypsin solution (Gibco) containing 100 units/ml DNAse 1 (Roche, Basel, Switzerland) 10μM Y27362 for 25 minutes at 37° with shaking and trituration. Dissociated cells were then washed 2x in DMEM and passed through a 40μm cell strainer before resuspension in ice-cold HBSS containing 1% BSA at a concentration of 1000 cells/μl. Cells were then filtered a second time and immediately used for library preparation. Library preparation by the NuSeq core at the Northwestern University Feinberg School of Medicine using the 10x Genomics Chromium platform. Libraries were then sequenced using a HiSeq4000 instrument at a depth of 50,000 reads per cell. single cell RNA-Seq