GSM9651350: KM_NTC_2_Totals; Mus musculus; RNA-Seq
Cells were then gently lifted, pelleted in PBS and  lysed in 500 µl ice-cold lysis buffer (15 mM Tris-HCl pH 7.5, 15 mM MgCl2, 150 mM NaCl, 1% Triton X-100, 0.05% Tween 20, 2% n-Dodecyl β Maltoside (Thermo Fisher Scientific, 89903), 0.5 mM DTT, 100 µg/ml cycloheximide, 1× cOmplete EDTA-free Protease Inhibitor Cocktail (Merck, 11836170001), 200 U/ml RiboLock RNase Inhibitor (Thermo Fisher Scientific, EO0381)) on ice for 5 min with agitation every minute. Lysates were centrifuged at 4°C for 5 min at 12,000 g and 500 µl supernatant was pipetted into a fresh 1.5 ml tube. For cytoplasmic RNA samples, RNA was extracted from 25 µl of lysate with 975 µl TRIzol, as per the manufacturer's instructions. A 20-µl aliquot of lysate was also added to 5 µl of 5× SDS gel-loading buffer for western blotting to check for depletion of p. The remaining lysate was digested with 5 µl Ambion RNase I (cloned, 100 U/µl; Thermo Fisher Scientific, AM2295) at 22°C for 15 min, with agitation at 600 rpm in a thermo-mixer. The digestion was stopped with 10 µl SUPERaseIn RNase Inhibitor (20 U/μl; Thermo Fisher Scientific, AM2696) and samples were loaded onto a 10–50% sucrose gradient, containing 15 mM Tris-HCl pH 7.5, 15 mM MgCl2, 150 mM NaCl, and 100 µg/ml cycloheximide, prepared using a BioComp gradient station and cooled to 4°C for at least 1 h prior to use. Samples were centrifuged in a Beckman XPN-90 Ultracentrifuge with an SW40Ti rotor at 38,000 rpm for 2 h at 4°C. Fractions of 0.5 ml were collected with a Biocomp gradient station and Gilson FC 203B fraction collector. Fractions containing the 80S ribosome peak were extracted with acid phenol:chloroform, followed by two chloroform washes. RNA was precipitated with 2 µl glycogen (Roche 10901393001), 300 mM NaOAc pH 5.2, and an equal volume of isopropanol overnight at -20 °C. RNA was pelleted by centrifugation at 12,000 g for 45 min at 4 °C. The supernatant was removed with a pipette, and the pellet was washed twice with 70% ethanol and dissolved in 10 µl RNase-free water. RNA was diluted with 10 µl 2× urea buffer (Thermo Fisher Scientific, LC6876), heated at 80°C for 90 s, placed immediately on ice, and then loaded onto a pre-run 15% TBE-urea gel (Thermo Fisher Scientific, EC68852BOX) and ran at 200 V for 1 h alongside custom 28 nt (AGCGUGUACUCCGAAGAGGAUCCAACGU) and 34 nt (GCAUUAACGCGAACUCGGCCUACAAUAGUGACGU) RNA markers. The gel was stained with 1× SYBR gold (Thermo Fisher Scientific, S11494) and imaged on a Typhoon FLA 7000. An image was printed to size to allow bands, inclusive of 28 nt and exclusive of 34 nt markers, to be cut from the gel, placed into a 1.5 ml RNA low-binding microcentrifuge tube, and crushed with a scalpel. RNA was eluted from the crushed gel pieces in 500 µl extraction buffer (300 mM NaOAc pH 5.2, 1 mM EDTA, 0.25% SDS) overnight at 16°C at 600 rpm in a thermo-mixer. Gel pieces were filtered out with a Costar Spin-X centrifuge tube filter (0.45 µm; Scientific Laboratory Supplies Ltd, 8163) and RNA was precipitated with 2 µl glycogen and 500 µl isopropanol overnight at -20 °C. Precipitated RNA was pelleted, washed, and dissolved in 13.5 µl RNase-free water, as above. To deplete rRNA the following was added to the 13.5 µl RNA; 5 µl hybridisation buffer (10 mM Tris-HCl pH7.5, 1 mM EDTA, 2 M NaCl), 1 µl RNasin Plus Ribonuclease Inhibitor (Promega N2615) and 0.5 µl biotinylated DNA oligo pool (100 uM total DNA with rRNA_depl_1 and rRNA_depl_2 at a 3:1 molar ratio compared to all other oligos), that had been denatured at 95 °C for 3min and then placed immediately on ice. The mix was incubated for 10min at 68 °C at 1250 rpm in a thermo- mixer and then allowed to cool slowly to room temp by turning off the thermo-mixer. rRNA was depleted with 160 µl Dynabeads MyOne Streptavidin C1 (Thermo Scientific, 65001) as per manufactures' instructions. The depleted non-RNA was precipitated with 2 µl glycogen and three times the volume of ethanol. Precipitated RNA was again pelleted, washed, and dissolved in 43 µl RNase-free water, as above. RNA was heated at 80°C for 90 s and immediately placed on ice before undergoing 5′ phosphorylation and 3′ dephosphorylation with 1 µl T4 PNK (NEB, M0201S), 5 µl 10× PNK buffer, and 1 µl SUPERaseIn RNase Inhibitor (20 U/μl) at 37°C for 35 min, with 5 µl 10 mM ATP added for the final 20 min. RNA was extracted with acid phenol:chloroform and isopropanol-precipitated as above. Purified RNA was input into the Bioscientific Nextflex small RNA v3 kit (NOVA-5132-06), using the alternative step F bead clean-up, 15 PCR cycles, and gel-extraction option. Cytoplasmic RNA samples were run on an Agilent TapeStation to check RNA integrity. RNA concentration and sample purity were measured on a NanoDrop spectrophotometer. rRNA was depleted from 1 µg cytoplasmic RNA with the RiboCop v2, and sequencing libraries were prepared from the rRNA-depleted RNA with the Corall Total RNA-Seq Library Prep Kit v1 (Lexogen, 095.96) using 13 PCR cycles. Final cytoplasmic RNA and RPF libraries were quantified on an Agilent TapeStation, with a High Sensitivity D1000 tape (Agilent), and sequenced single-end on an Illumina NextSeq500 instrument with a 75 cycles high-output kit.