title: GSM8717419: AstrocyteRiboTag_male_2h_ischemia_6h_reperfusion_ipsi_rep3; Mus musculus; RNA-Seq
library_construction_protocol: MCA territories were dissected, then homogenized by hemisphere in a 7 mL glass Dounce homogenizer (12–15 strokes) in 1× HBSS containing Ca2+/Mg2+ (Gibco, Waltham, MA, USA) and supplemented with HEPES. Homogenates were centrifuged at 2000× g for 10 min at 4 °C. Pellets were resuspended in 18% dextran (from Leuconostoc spp., Mr~70,000, MilliporeSigma, Burlington, MA, USA), then centrifuged at 10,000× g for 15 min at 4 °C. Myelin-cleared pellets were resuspended in 1% bovine serum albumin, then filtered through a 20 μm nylon mesh. Microvessels caught in the mesh were retrieved and centrifuged at 2000× g for 10 min. For RiboTag immunoprecipitation, solutions were supplemented with 100 µg/mL cycloheximide and 200 U/mL RNAsin. For RiboTag IP, all steps were performed in 4 °C and RNase-free (RNaseZAP, ThermoFisher, Waltham, MA, USA) conditions. Microvessels were lysed in freshly prepared lysis buffer (150 mM KCl, 20 mM HEPES [pH 7.4]), 5 mM MgCl2, 1 mM DTT, 1% NP-40, 100 µg/mL cycloheximide, and 30 mM DHPC [1,2-dicaproyl-sn-glycero-3-phosphocholine, 07:0]) supplemented with 200 U/mL RNAsin (ProMega, Madison, WI, USA) and cOmplete protease inhibitor (MilliporeSigma). Following homogenization using a handheld pestle motor mixer, lysates were centrifuged at 18,000× g at 4 °C for 10 min. IP was performed by incubating the soluble fraction with magnetic bead-conjugated anti-GFP antibody (#67090, Cell Signaling Technology, Danvers, MA, USA) or magnetic bead-conjugated control IgG (#5873, Cell Signaling Technology) overnight (~16 h) with end-over-end rotation. Following magnetic separation from the supernatant (DynaMag-2, ThermoFisher), beads were washed three times in a high-salt (300 mM KCl) lysis buffer supplemented with 200 U/mL RNAsin. Unpurified RNA was collected from the magnetic beads using the TriZOL reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. Total mRNA (in ultrapure water) following RiboTag IP was quantified using the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Strand-specific RNA sequencing was performed by Maryland Genomics in NovaSeq S4 flow cells (NovaSeq6000, Illumina, San Diego, CA, USA) with 100 bp paired-end reads, with 67 ± 18 million (mean ± SD) reads per sample.