title: GSM5268226: RNAseq2; Rattus norvegicus; RNA-Seq
library_construction_protocol: Five P5 rat brains were homogenized in RNA Granule Buffer (20 mM TRIS-HCl pH 7.4, 150 mM NaCl, 2.5 mM MgCl2 supplemented with 1 mM DTT , 1 mM EGTA and Roche EDTA-free protease inhibitor in the presence or absence of 100 ug/ml cycloheximide. This homogenate was used for RNA-SEQ libraries. For Footprinting libraries, homogenate was centrifuged 15 minutes in a Thermo Scientific T865 fixed angle rotor at 9200 rpm (6117 RCF) at 4˚C to spin down cellular debris. The supernatant was treated with 1% IGEPAL CA-630 for 5 minutes at 4˚C on a rocker. The sample was then loaded onto a 2 ml 60% sucrose] cushion (dissolved in supplemented RNA Granule Buffer) in a Sorvall 36 ml tube, filled to top with additional RNA Granule Buffer and centrifuged for 2 hours in a Thermo Scientific AH-629 swing-bucket rotor at 28500 rpm (56660 RCF) at 4˚C to achieve the polysome pellet. The pellet was re-suspended in RNA Granule Buffer, gently dounced and loaded over a 15-60% linear sucrose gradient (gradient was made with RNA Granule Buffer) and centrifuged for 45 minutes at 28500 rpm (56660 RCF) at 4˚C in an AH-629 swing bucket rotor. Fractions of 3.5 ml were then collected from the top, and the remaining pellet was rinsed once and then resuspended using RNA Granule Buffer. For treatments with salt, the suspension was incubated with 400 mM NaCl for 10 minutes at 4˚C on a rocker and then NaCl-free RNA granule buffer was added for a final concentration of 150 mM NaCl. The sample was then treated with 100 U of RNase I [100 U/µl; Ambion AM2294, Thermo Fisher] for 30 minutes at 4˚C on a rocker. The nuclease was quenched with 100 U of SuperaseIN [20 U/µl; Invitrogen #AM2696, Thermo Fisher], and the samples were re-run on a fresh 15-60% sucrose gradient to separate mononsomes. Either Fraction 2 or homoegnates (for RNA-SEQ) was precipitated overnight at -20 ˚C by adding 7 ml of chilled 100% ethanol. Precipitates were centrifuged at 4,444 x g at 4 °C for 60 min, the isopropanol taken off, the pellet air-dried, and re-suspended in 10 mM Tris-HCl pH 7. We then proceeded with a standard hot phenol-acid extraction (Acid Phenol:Chloroform mix 125:24:1, Invitrogen, AM9722). SI All samples were depleted of ribosomal RNA using the Ribo-Zero Gold (Human/Mouse/Rat) Kit (Illumina) (replicates 1-3 and cycloheximide treated ) or the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat)(E6350, NEB) (replicates 4-5 and no salt treatment) The footprint samples were size-selected using the 17 and 34 nt markers as a guide on a 15% TBE-Urea polyacrylamide gel (Thermo Scientific), while total RNA samples were randomly heat fragmented by incubating at 95 °C in an alkaline fragmentation solution (50 mM NaCO3 pH 9.2, 2 mM EDTA) for 40 min, yielding comparably sized fragments to footprints. All samples were de-phosphorylated using PNK (T4, NEB). The quality and concentration of samples was assessed by running an Agilent Small RNA chip (Agilent Technologies), and sequencing libraries were generated using the NEXTflex™ Small RNA Sequencing Kit v3 (PerkinElmer, NOVA-5132-06), according to the manufacturer's instructions. 