Dysregulated DNA Methylation in Abca4-/- Retinal Pigment Epithelium: Insights into Early Stage of Stargardt Disease
Dysregulated DNA Methylation in Abca4-/- Retinal Pigment Epithelium: Insights into Early Stage of Stargardt Disease
Mice were euthanized by cervical dislocation, and eyes were harvested for obtaining RPE/eyecup separated from neural retina. In 1x phosphate buffer saline (PBS, pH 7.4), the anterior segment of the eye was removed, followed by the separation of the neural retina from the eyecup. RPE/Eyecups were stored in a −80 °C freezer until further processing. DNA was extracted from mice, one RPE/eyecup per sample, using the Dneasy Blood & Tissue Kit (Qiagen, Hilden, Germany, Cat# 69506). DNA was eluted in 50 μL AE Buffer. The concentration was measured with a Qubit instrument (Life Technologies, Carlsbad, CA, USA), and 100 ng of genomic DNA was treated with RNAse enzyme at 37 °C for 30 min to remove contaminating RNA. RRBS libraries were prepared by a Zymo-Seq RRBS Library kit (Zymo research, Irvine, CA, USA, Cat# D5460). The libraries were subjected to size selection using magnetic AMPure XP beads (Beckman Coulter Life Sciences, Indianapolis, IN, USA) to enrich DNA fragments of the desired size. The quality control of the final libraries (size-selected and barcoded) was performed using an Agilent TapStation 4200 (D1000, D5000; Agilent Technologies, Santa Clara, CA, USA).