AGO CLIP reveals an activated network for acute regulation of brain glutamate homeostasis in ischemic stroke [dataset 5]

Cortical cultures treated with OGD (oxygen glucose deprivation) in the presence of miR-29 expressing lenti-virus were analyzed by RNAseq to assess differences in miR-29 target gene expression. DIV6 cortical cultures were infected with virus expressing miR-29 or empty control virus at (MOI=1) two times, for total MOI of 2. On DIV14, cultures were first rinsed twice with warm OGD buffer (125 mM NaCl, 5 mM KCl, 1.2 mM Na2PO4, 26 mM NaHCO3, 1.8 mM CaCl2, 0.9 mM MgCl2, 10 mM Hepes, pH 7.4) plus 10 mM glucose and incubated for 30 min with the same rinsing buffer. The cultures were then rinsed with OGD buffer twice and transferred to an OGD chamber (Billups-Rothenberg, San Diego, CA) and flushed with anoxic gas (95% N2 and 5% CO2) at 21 L/min for 5 min. The chamber was then sealed and placed into 37°C incubator for 3 hrs. Controls (sham) were rinsed with OGD buffer but incubated in a normoxia conditions. After OGD treatment, the cultures were transferred to normal culture medium and incubated for 4 hrs, cells harvested and RNA extracted for library construction. Each set of biological replicates represent cultures dissected on distinct days. Complete protocol detailed in referenced publication.