title: E12.5 6.8 D
organism: Mus musculus
description: Protocols: C57Bl6 mouse embryos were collected at E11.5 and E12.5. The embryos were dissected in PBS and heads were dissected, followed by embedding in OCT medium (VWR) and snap freezing on dry-ice and storage at -80°C prior to sectioning. The embedded heads 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-12-17T17:08:36Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-08-10T16:23:13Z
attributes.tag: External Id
attributes.value: SAMEA7171753
attributes.tag: INSDC center name
attributes.value: UCL Great Ormond Street Institute of Child Health
attributes.tag: INSDC first public
attributes.value: 2020-12-17T17:08:36Z
attributes.tag: INSDC last update
attributes.value: 2020-08-10T16:23:13Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9427:E12.5 6.8 D
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryonic day 12.5
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: 6.8
attributes.tag: organism part
attributes.value: eye primordium
attributes.tag: sample name
attributes.value: E-MTAB-9427:E12.5 6.8 D
attributes.tag: sampling site
attributes.value: dorsal optic cup
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: not available
attributes.tag: strain
attributes.value: C57BL/6