GSM2057925: Day 1 control; Xenopus laevis; RNA-Seq
Both left and right eyes were harvested separately.  Retinas were isolated by removing the lens, then peeling off and discarding the retinal pigment epithelial layer. Freshly dissected retinas were immersed in ice-cold lysis buffer (20 mM HEPES KOH, pH 7.4, 5 mM MgCl2, 150 mM KCl) to which was added freshly prepared 100 μg/ml cycloheximide, 0.5 mM DTT, protease inhibitors (Roche Mini Complete, EDTA-Free) and 40 U/ml recombinant Rnasin (Promega).  We isolated total RNA from the retina or used the translation ribosomal affinity purification (TRAP) protocol to isolate and purify the RNAs specific to the RGCs (described in Watson et al., 2012; DOI 10.1002/dvdy.23880).  To immunoprecipitate ribosomes and associated RNAs, we used equal amounts of anti-eGFP antibodies (19C8 and 19F7; Memorial Sloan-Kettering Monoclonal Antibody Facility) at a concentration of 100 µg of total anti-eGFP antibody per 375 µl of Dynal Protein G-magnetic beads (Life Technologies). To obtain a minimum of 120 ng/sample of purified TRAP-isolated mRNAs, equal quantities from each of the three independent biological replicates were combined to generate a single pooled sample.  RNA samples were then sent to the JHMI Deep Sequencing Core for RNA-Seq cDNA library construction using poly(A) selection, with standard Illumina protocols for HiSeq 2000.
GEO Accession
GSM2057925