GSM8950652: VHL treated APPPS1-21 cortex 1; Mus musculus; RNA-Seq
We followed Heiman et al 2014 for extraction of RNA for TRAPSeq. To capture eGFP+ polysomal complexes in astrocytes for TRAPseq, an anti-GFP affinity matrix (AM) was prepared prior to homogenization of cortical tissue using a previously published protocol(27). Streptavidin MyOne T1 Dynabeads were resuspended, and 450 µL/purification was added to the AM tube. Beads were collected by a magnet and were washed with 1X PBS. 180 µL/purification of biotinylated protein L was then added to the beads and the mixture was incubated on a tube rotator for 35 minutes. The protein L coated beads were captured on a magnet and washed 5 times with 1X PBS containing 3% (w/v) IgG and protease free BSA. 75 µg of anti-GFP antibodies (Htz-GFP-19F7 and Htz-GFP-19C819F7 obtained from Memorial Sloan Kettering Cancer Center Antibody & Bioresource Core Facility) in low salt buffer (20 mM HEPES KOH, 150 mM KCl, 10 mM MgCl2, and 1% (vol/vol) NP-40 in RNase-free water) were then added to the bead mixture and was incubated on a tube rotator for 1 hour. After antibody binding, the final AM was washed 3x with low salt buffer. After washing, the AM was resuspended in a volume of low salt buffer such that each IP received an aliquot of 200 μL. Following AM preparation, flash frozen mouse cortices (washed in dissection buffer before freezing) were homogenized in 1.5 mL tissue lysis buffer (20 mM HEPES KOH, 150 mM KCl, 10 mM MgCl2, EDTA-free protease inhibitor tablet, 0.5 mM DTT, 100 µg/ml cycloheximide in RNase-free water) in a glass tube with an electric homogenizer (Glas-Col #099CK5424) at 900 RPM with 15 full strokes. Lysates were transferred into prechilled microcentrifuge tubes on ice. Post-nuclear supernatants were prepared (S2) by centrifugation at 4°C for 10 minutes at 2000g. S2 was transferred to a new, prechilled microcentrifuge tube on ice. 1/9 sample volume of 10% NP-40 to S2 (final concentration 1%) was added and mixed gently by inverting the tube. 1/9 sample volume of 300 mM DHPC was added to S2 (final concentration 30 mM) and mixed gently by inverting the tube and the mixture was incubated on ice for 5 minutes. Post-mitochondrial supernatants (S20) were prepared by centrifugation at 4°C for 10 minutes at 20,000 xg. S20 was transferred to a new, prechilled microcentrifuge tube. 200 uL of freshly resuspended AM was added to each S20 sample. Beads complexes were washed 4x with high salt buffer (20 mM HERPES, 350 mM KCl, 10 mM MgCl, 1% NP-40 in RNase-free water). RNA was isolated from beads using 100 μL of Nanoprep lysis buffer (from Stratagene Absolutely RNA Nanoprep kit). RNA cleanup was performed according to the kit. Quality of RNA isolated from TRAP purifications was assessed using Agilent Bioanalyzer. All RNA used for downstream sequencing had a RIN score above 7. mRNA libraries were generated from TRAP-purified RNA using the Illumina Stranded mRNA prep kit. Prepared libraries were sequenced on an Illumina HighSeq 4000.