GSM7968157: NPM_WT_4_RPFs; Mus musculus; OTHER
Intestinal crypts were isolated from the proximal part of the small intestine following removal of the villi. To achieve this, intestines were flushed with PBS, cut lengthwise and incubated at 37°C for 7 minutes with regular agitation in RPMI 1640 media (Thermo Fisher Scientific, 21875059) containing 10 mmol/L EDTA and 200 μg/mL cycloheximide. The tissue was then transferred to ice-cold PBS with 10 mmol/L EDTA and 200 μg/mL cycloheximide for 7 minutes with regular agitation. Tissue was then discarded and crypts pelleted by centrifugation for 5min at 350g at 4oC and snap froze in liquid N2 and stored at -80°C. Pellets were placed at -20°C for 1h and then on ice for 10min, prior to lysis with 500 µl ice cold lysis buffer (15 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 300 mM NaCl, 1% Triton X100, 0.05% Tween20, 2% n Dodecyl β Maltoside (Thermo Fisher 89903), 0.5 mM DDT, 100 µg/ml cycloheximide, 1X cOmplete EDTA-free Protease Inhibitor Cocktail (Merck 11836170001), 200 U/ml RiboLock RNase Inhibitor (Thermo Fisher Scientific, EO0381)) on ice for 5 min while titrating through a 21G needle with a 1ml syringe. Lysates were centrifuged at 4°C for 5 min at 12,000g and 500 µl supernatant pipetted into a fresh 1.5 ml tube. For cytoplasmic RNA sample, RNA was extracted from 25 µl lysate with 975 µl TRIzol, as per manufactures' instructions. 20 µl lysate was also added to 5 µl 5X SDS gel loading buffer for Western blot to check for depletion of eIF4A1 or eIF4A2 in the knock-out samples. Remaining lysate was digested with 10 µl Ambion RNase I cloned, 100 U/µL (Thermo Fisher Scientific, AM2295) at 22°C for 15 min at 600 rpm in a thermo mixer. The digestion was stopped with 20 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, 300 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. Purified RNA was dissolved in 10 µl RNase free water and diluted with 10 µl 2X urea buffer (Thermo Fisher Scientific, LC6876), heated at 80°C for 90 sec, placed immediately on ice and then loaded onto a pre-run 15% TBE-Urea gel (Thermo Fisher Scientific, EC68852BOX) and ran at 200V for 1h alongside 28nt (AGCGUGUACUCCGAAGAGGAUCCAACGU) and 34nt (GCAUUAACGCGAACUCGGCCUACAAUAGUGACGU) RNA markers. Gel was stained with 1X SYBR gold (Thermo Fisher Scientific, S11494) and imaged on a Typhoon FLA 7000. 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-bind tube and crushed with 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 Filters 0.45 µm (SLS 8163) and RNA was precipitated with 2 µl glycogen and 500 µl isopropanol overnight at -20°C. Precipitated RNA was purified and dissolved in 43 µl RNase free water. RPF RNA was not rRNA depleted. RNA was heated at 80°C for 90 sec and immediately placed on ice before undergoing 5′ phosphorylation and 3′ dephosphorylation with 1 µl T4 PNK (NEB, M0201S), 5 µl 10X PNK buffer and 1 µl SUPERaseIn RNase Inhibitor (20 U/μL) (Thermo Fisher Scientific, AM2696) at 37 °C for 35 min, with 5µl 10mM ATP added for final 20 min. RNA was extracted with acid phenol chloroform and isopropanol precipitated. Purified RNA was quantified on a qubit with the high-sensitivity RNA kit (Q32852) and ran on an agilent bioanalyser to check for correct length. 10 ng RNA was input into the Bioscientific Nextflex small RNA v3 kit (NOVA-5132-06), using the alternative step F bead clean up, 11 PCR cycles and gel extraction option. Cytoplamsic RNA samples were run on an agilent tapestation to check for integrity and measured on a nanodrop to quantify and check for purity. 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 (Lexogen Cat. 095.96) with 13 PCR cycles. Final cytoplamsic RNA and RPF libraries were quantified on an agilent tapestation with a High Sensitivity D1000 tape and sequenced single-end on an Illumina NextSeq500 with a 75 cycles high output kit.