NextSeq 500 sequencing; RNA-seq of peri-infarct cortex of the rat dMCAO model of ischemic stroke treated with AAV7-hMANF or AAV7-GFP
RNA-seq of peri-infarct cortex of the rat dMCAO model of ischemic stroke treated with AAV7-hMANF or AAV7-GFP
Animals were anesthetized with i.p. pentobarbital and perfused transcardially with 0.9% saline solution. The brains were frozen in isopentane on chilled on dry ice. 1 mm thick sections were cut from the brains and punches of cerebral cortex adjacent to the infarct (the peri-infarct cortex) were collected from positions A/P -1.0 to +0.0 and A/P +1.0 to +2.0. Male Sprague-Dawley rats (280-320g in 1st study, 200-280g in 3rd study), were anesthetized with intra-peritoneal chloral hydrate injection (0.4 g/kg) and underwent a 60 minute dMCAo surgery. On day 2 after distal middle cerebral artery occlusion surgery the animals were anesthetized with isoflurane and attached to a stereotaxic frame. dsAAV7s were injected intracerebrally into two subcortical sites with the following stereotaxic coordinates: site 1 A/P +1.6; L/M +2.2; D/V −5.0 (from the surface of the skull) and site 2 A/P −0.4; L/M +4.0; D/V −5.0. Two and a half microliters of AAV7-GFP (titer 1.1 × 1013 vg/mL) or AAV7-hMANF (8.1 × 1012 vg/mL) was injected at a speed of 1 µl/min using a 10 μl Hamilton syringe with a 30 G blunt needle. The needle was kept in place for 2 min after the injection. RNA was extracted with Trizol reagent and treated with DNase (#1906, Ambion) rRNA was removed and the sequencing libraries were prepared using Illuminan TruSeq Stranded Total RNA Library Prep Kit with Rbo-Zero Human/Mouse/Rat