NextSeq 500 paired end sequencing; RNA-seq of the developing human eye (Day 41-44 of embryonic development) comparing gene expression in ventral optic fissure margins with dorsal optic cup
Human embryonic eyes at CS17 and CS18 were dissected and the eye along with surrounding periocular mesenchyme was embedded in OCT medium (VWR) and snap frozen on dry ice and stored at -80°C. The embedded eyes were sectioned in the parasaggital plane, i.e. perpendicular to the optic fissure at a thickness of 20µm at -20°C, sections were collected on PEN (polyethylene naphthalate) membrane coated slides (Thermo Fisher LCM0522) and stored -80°C. Slides The eye was orientated and optic fissure identified, under bright field illumination on a laser capture microdissection microscope (P.A.L.M microbeam). The region of interest containing the fissure margins was identified, outlined and cut out using a UV laser and catapulted with the laser into the cap of a collection tube (Zeiss 415190-9211-000). Fissure margin regions were collected along the length of the fissure beginning with the first section in which both margins could be clearly visualized and ending with the last section before the optic nerve head became visible.  The fissure margins of the two eyes of each embryo were pooled in the same collection tube. Similarly, a control region was cut out of the dorsal optic cup and pooled using a fresh collection tube. Lysis buffer (Qiagen RNeasy Micro kit, 74004) was added to the collection tube, the caps were closed and the tube was inverted lyse the tissue and prevent RNA degradation,frozen on dry ice and stored at -80°C. Total RNA was extracted using standard protocols, and treated with DNAseI (Qiagen). RNA quality and concentration were determined using the Tapestation (Agilent Technologies).  RNA samples were amplified and cDNA libraries prepared using the SMART-Seq v4 Ultra Low Input RNA (Clontech Laboratories) and Nextera XT DNA Library Preparation (Illumina) kits and cDNA quality was assessed using Qubit dsDNA HS Assay kit and Qubit 2.0 Fluorometer (ThermoFisher).