GSM9035737: DBA/2J_Pool 1; Mus musculus; RNA-Seq
Mouse eyes were enucleated and immediately placed in individual 1.5 ml tubes containing 0.5 mL of RNAprotect® Tissue Reagent (Qiagen, 76104) and stored at 4 °C until dissection. For dissection, the anterior and posterior chambers were separated. The anterior segment was divided into four quadrants, and the iris and ciliary body were carefully removed under a dissection microscope to avoid damaging the trabecular meshwork (TM). The iridocorneal angle was carefully dissected, with trimming of non-target tissues to minimize contamination. Iridocorneal angles from three individual mice were pooled in a 1.5 mL tube containing 100 µL of RNAprotect and stored at 4 °C until RNA extraction. RNA isolation was performed using the RNeasy® Plus Micro Kit (Qiagen,74034) according to the manufacturer's protocol, with some modifications. Briefly, RNAprotect was removed, and tissues were lysed in 350 µL Buffer RLT Plus supplemented with 1% β-mercaptoethanol and Molecular Grinding Resin (Gbiosciences, 786038). Tissues were homogenized using motorized pestles for 1 min and passed through a Qiashredder column (Qiagen, 79656) by centrifugation at 13,000 rpm for 1 min. The lysate was transferred to a gDNA Eliminator spin column and centrifuged at ≥8000 × g for 30 s. An equal volume of 70% ethanol was added to the flow-through, mixed well, and the mixture was loaded onto an RNeasy MinElute spin column. After centrifugation at ≥8000 × g for 15 s, the column was sequentially washed with 700 µL Buffer RW1, 500 µL Buffer RPE, and 500 µL of 80% ethanol (repeated three times), each followed by centrifugation at ≥8000 × g. The column was then centrifuged at full speed for 5 min with the lid open to remove residual ethanol. RNA was eluted with 14–22 µL RNase-free water and collected by centrifugation at full speed for 1 min. RNA concentration and purity were measured using a NanoDrop spectrophotometer (DS-11+ spectrophotometer, DeNovix). The Sequencing and Genomics Technologies Core Facility at Duke University performed RNA-sequencing. The SGT enriched mRNA from total RNA and reversed transcribed into cDNA to build sequencing libraries using the Kapa mRNA HyperPrep Kit from Roche (Code: KK8581). Libraries were pooled to equimolar concentration and sequenced on the NovaSeq X Plus 1.5B lane to produce 100 bp paired-end reads targeting 50M reads/sample