GSM5051864: I12,D1_WT_IP; Mus musculus; RNA-Seq
Trap-Seq was carried out as described in (Heiman et al., 2014). Briefly, striata from WT (n=6) and FXS (n=5) model mice expressing the EGFP-tagged Rpl10a protein in dopamine receptor Drd1-expressing medium spiny neurons were lysed by dounce homogenization (40 strokes) in 25 mM Hepes-HCl (pH 7.3), 150 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 100 μgml−1 Cycloheximide, 10 μlml−1 RNasin (Promega, Madison, WI) and 10 μlml−1 Superase-In (Life Technologies) RNase inhibitors, and 1X Halt protease/phosphatase inhibitor on ice. Nuclei were pelleted by centrifuging the lysates at 2000g. NP-40 (Sigma) and DHPC (Avanti Polar Lipids, Alabaster, AL) were each added to the supernatant to a final concentration of 1%, following which the lysates were centrifuged at 20,000g in order to pellet insoluble membranes. A small aliquot of the supernatant was saved for RNA-Seq (input). EGFP- tagged ribosomal protein L10a was precipitated by incubating the remaining supernatant overnight (4◦C) with 100 μg of monoclonal anti-EGFP antibodies (50 μg each of clones 19C8 and 19F7) bound to biotinylated-Protein L (Pierce, Thermo Fisher, Waltham, MA) coated streptavidin-conjugated magnetic beads (Life Technologies). The magnetic beads were then washed four times in high-salt buffer consisting of 10 mM Hepes-HCl (pH 7.3), 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5 mM DTT, 100 μgml−1 Cycloheximide, and RNasin and Superase-In RNase inhibitors (Promega). Bound RNA was eluted and purified using the Absolutely RNA Nanoprep kit (Agilent, Santa Clara, CA). Sequencing libraries (non strand-specific) from the IP and input RNA were prepared using the Nugen Ovation Trio Low Input RNA kit at the NYUMC Genome Technology Center. Libraries were sequenced on the Illumina NovaSeq S1 100 Cycle Flow Cell to generate 50-cycle paired-end reads. All pulldowns and sequencing were carried out in a single batch.