AGO CLIP reveals an activated network for acute regulation of brain glutamate homeostasis in ischemic stroke [dataset 1]

Adult mouse cortical tissues after MCAO or sham surgery were analyzed by Argonaute HITS-CLIP to assess differences in transcriptome-wide AGO binding patterns in I/R injury model of stroke. Mice were anesthetized briefly with 1.5–2% isoflurane. A heat-blunted nylon suture (6/0) was inserted into the right external carotid artery and advanced until it obstructed the MCA together with the ligation of the common carotid artery for 35 min. Regional cerebral blood flow (CBF, bregma coordinates: 2-mm posterior, 5-mm lateral) was continuously recorded by transcranial laser Doppler flowmetry from the induction of ischemia until 30 min after reperfusion. Surgery success was determined by monitoring regional cerebral blood flow (CBF, bregma coordinates: 2-mm posterior, 5-mm lateral) using transcranial laser Doppler flowmetry, and required a residual CBF <15%, followed by >80% recovery within 10 min of reperfusion. Following MCAO, mice were placed in temperature-controlled recovery cages for 3 h to prevent post-surgery hypothermia and sacrificed. Sham animals were similarly anestheized, arteries exposed for surgical time period without filament insertion. Cortical tissues from ipsilateral or contralateral hemispheres of MCAO or sham animals were dissected, UV-irradiated and analyzed by CLIP protocol. For each CLIP sample, high and low molecular weight AGO:RNA complexes (~110-130 kDa, Supplementary figure 1B) were pooled for library construction. Each set of biological replicates were prepared separately by AGO-CLIP and consist of representative samples from a distinct day of surgery. Complete protocol detailed in referenced publication.