Name: GSM7113190
Instrument: NextSeq 550
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: RNA from the hippocampus of one hemisphere of Tam-induced Camk2a-cre/ERT2+;NuTRAP+ mice was isolated via the TRAP method (Input, Negative, and Positive fractions). Tissue was minced into small pieces and homogenized in 100 ul ice-cold homogenization buffer (50 mM Tris, pH 7.4; 12 mM MgCl2; 100 mM KCl; 1% NP-40; 1 mg/mL sodium heparin; 1 mM DTT) supplemented with 100 ug/mL cycloheximide (#C4859-1ML, Millipore Sigma), 200 units/mL RNaseOUT™ Recombinant Ribonuclease Inhibitor (#10777019; Thermofisher), and 1X cOmplete™, EDTA-free Protease Inhibitor Cocktail (#11836170001; Millipore Sigma) with a Kimble pellet pestle cordless motor with one pulse of 10 sec/each on ice. An additional 300 uL of homogenization buffer was added and homogenized with pellet pestle cordless motor with one pulse of 10 sec/each (on ice). Volume was taken up to 1.5 mL with homogenization buffer and centrifuged at 12,000 x g for 10 minutes at 4°C. After centrifugation, 100 uL of the supernatant was saved as input. The remaining supernatant was transferred to a 2 mL round-bottom tube and incubated with 5 ug/uL of anti-GFP antibody (ab290; Abcam) at 4°C with end-over-end rotation for one hour. Dynabeads™ Protein G for Immunoprecipitation (#10003D; Thermofisher) were washed three times in 1 mL ice-cold low-salt wash buffer (50mM Tris, pH 7.5; 12mM MgCl2; 100mM KCl; 1% NP-40; 100μg/ml cycloheximide; 1mM DTT). After removal of the last wash, the homogenate/antibody mixture was transferred to the 2 mL round-bottom tube containing the washed Protein-G Dynabeads and incubated at 4°C with end-over-end rotation for an additional two hours. Magnetic beads were collected using a DynaMag-2 magnet and the unbound- ribosomes and associated RNA saved as the “negative” fraction. Beads were then washed three times with 1 mL of high-salt wash buffer (50mM Tris, pH 7.5; 12mM MgCl2; 300mM KCl; 1% NP-40; 100μg/ml cycloheximide; 2mM DTT). Following the last wash, 350 uL of Buffer RLT (Qiagen) supplemented with 3.5 uL 2-β mercaptoethanol was added directly to the beads and incubated with mixing on a ThermoMixer (Eppendorf) for 10 minutes at room temperature. The beads were magnetically separated and the supernatant containing the target bead-bound ribosomes and associated RNA was transferred to a new tube. 350 uL of 100% ethanol was added to the tube (“positive” fraction) and then loaded onto an RNeasy MinElute column. RNA was isolated using RNeasy Mini Kit (#74104, Qiagen), according to manufacturer's instructions. RNA was quantified with a Nanodrop 2000c spectrophotometer (Thermofisher Scientific) and its quality assessed by HSRNA screentape with a 2200 Tapestation analyzer (Agilent Technologies) The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used on 100 ng of total RNA for the preparation of strand-specific sequencing libraries from each TRAP-isolated RNA sample (input, negative fraction, and positive fraction), according to manufacturer's instructions. Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, the addition of a single 'A' base, and finally ligation of adapters. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HS DNA ScreenTape (#5067-5582; Agilent Technologies) and libraries were quantified by Qubit dsDNA HS Assay Kit (ThermoFisher Scientific). The libraries for each sample were pooled at 4 nM concentration and sequenced using an Illumina NextSeq 6000 system (PE75bp) at the Oklahoma Medical Research Foundation Genomics Facility.