GSM8486777: Myc_day4_1_Totals; Mus musculus; OTHER
Livers were dissected on surfaces cleaned and treated with RNase Zap to reduce RNAase exposure. Livers were rapidly dissected and snap frozen as 5x5x5mm fragments in liquid nitrogen. Livers were ground to powder using a mortar and pestle with the CryoGrinder system (OPS Diagnostics, Lebanon, NJ, USA) to ensure the samples were kept frozen. Roughly 200 mg tissue aliquots were stored in 1.5 ml tubes at -80 ºC until required.Ground liver tissue was poured directly from a 1.5 ml tube on dry ice into 900 µl ice cold lysis buffer (15 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 150 mM NaCl, 1% Triton X100, 0.05% Tween 20, 2% n Dodecyl β Maltoside (Thermo Fisher 89903), 0.5 mM DDT, 100 µg/ml cycloheximide, 1X cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Merck 11836170001),  1x Protease Inhibitor Cocktail (Sigma P9599-5ml), 5mM Sodium Fluoride, 500 U/ml RiboLock RNase Inhibitor (Thermo Fisher Scientific, EO0381), 25 U/ml Turbo DNase (Thermo Fisher Scientific, AM2239) on ice and immediately mixed together and placed on ice for 10 min, while being inverted every 2 minutes to maximise the dispersal and exposure of ground tissue to lysis buffer. Lysates were centrifuged at 4°C for 5 min at 16,000g and 800 µl supernatant pipetted into a fresh 1.5 ml tube on ice. For cytoplasmic RNA sample, RNA was extracted from 40 µl lysate with 1ml TRIzol, as per manufactures' instructions. 600µl lysate was digested with 1µl Ambion RNase I cloned, 100 U/µL (Thermo Fisher Scientific, AM2295), 1µl MNase (1:20 diln; New England Biolabs # 0247S) with 3.6µl 0.25M CaCL2, at 22°C for 15 min at 650 rpm in a thermo mixer. The digestion was stopped with 5 µl SUPERaseIn RNase Inhibitor (20 U/μL) (Thermo Fisher Scientific, AM2696) and 26µl 0.5M EGTA (to stop RNase 1 and MNase respectively). The 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 with a BioComp gradient station and cooled to 4°C for at least 1 h. Samples were centrifuged in a Beckman XPN-90 Ultracentrifuge with an SW40ti rotor at 38,000 rpm for 2 h at 4°C. 1 ml fractions were collected with a Biocomp gradient station and Gilson FC 203B fraction collector. Fractions pertaining to the 80S peak were extracted with acid phenol chloroform, followed by two chloroform washes and 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× TBE Urea sample 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 RNase free disposable pestle. 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 again pelleted, washed, and dissolved in 13.5 µl RNase-free water, as above, and underwent rRNA depletion as follows. To the 13.5 µl RNA was added, 5 µl hybridisation buffer (10mM Tris-HCl pH7.5, 1mM EDTA, 2M 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. This mix was incubated at 68 °C for 10 min at 1250 rpm in a thermo mixer and then allowed to cool slowly to room temp by turning off the thermomixer. rRNA was depleted with 160 µl Dynabeads MyOne Streptavidin C1 (Thermo Scientific, 65001) as per manufactures' instructions and depleted RNA was precipitated with 2 µl glycogen and ethanol. Precipitated RNA was again pelleted and washed as above and then dissolved in 43 µl RNase-free water and heated at 80 °C for 90 s and immediately placed on ice. RNA then underwent 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 quantified on a qubit with the high-sensitivity RNA kit (Q32852) and 5 ng was input into the Bioo Scientific Nextflex small RNA v3 kit (NOVA-5132-06), using the alternative step F bead clean-up, 14 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 ScreenTape (Agilent 5067-5584), and sequenced single-end on an Illumina NextSeq500 instrument with a 75 cycles high-output kit.