GSM4568887: RR927_m6Am_mouse_adult_Testes_input_HET; Mus musculus; OTHER
Total RNA from biological triplicates of 2 months-old wildtype (WT) and Pcif1 KO mice were used for the experiment with mouse brain, spleen and testis samples. The tissues were snap-frozen in liquid nitrogen. Total RNA was isolated from tissues using TRIzol (Thermo Fisher Scientific) according to manufacturer instructions. For ribosomal profiling brains from three adult wildtype (WT) controls and three Pcif1 KO mice were isolated and snap-frozen in liquid nitrogen. Frozen brain (~400 mg) was homogenized in 1300 µL (3x weight of the tissue in miligrams) of ice cold polysome lysis buffer (20 mM Tris?HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 5 mM DTT, 100 ?g/ml Cycloheximide, 40 U/ml RNasin plus, (Promega), Complete EDTA-free protease inhibitor (Thermo Scientific), 1% TritonX?100, 0.5% Sodium deoxycholate) using a glass homogenizer (6-8 strokes). The lysate was transferred to a 1.5 mL Eppendorf tube and incubated on ice for 10 minutes with intermittent mixing. The lysate was centrifuged at 3000 rpm for 3 minutes at 4°C. Supernatant was carefully collected in a new 1.5 mL Eppendorf tube. The OD260 of the lysate was measured with a Nanodrop instrument and approximately 15 OD260 (which correspond to 100 µL of lysate), was used for ribosome footprint preparation, and 50 µL was directly transferred to TriZol (Ambion) for total input RNA extraction. The remaining lysate was divided in aliquots of 150 µL and was snap frozen in liquid nitrogen and stored in -80°C freezer for later use. For ribosome footprint preparation, 100 µL of the supernatant was treated with 2.5 µL (5 units) of Turbo-DNase (Ambion) and 6.5 µL (650 units) of RNase I at 23°C for 45 minutes with rotation at 300rmp in a thermoblock. In meantime, the s-400 HR columns (GE healthcare) were washed three times with 700 µL polysome buffer (20 mM Tris?HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 5 mM DTT, 100 ?g/mL Cycloheximide, 40U/mL RNasin plus, (Promega), Complete EDTA-free protease inhibitor (Thermo Scientific)) plus 20U/mL SUPERaseIn (Ambion). During each wash columns with polysome buffer were spun at 2000rpm for 1 minute in a cold centrifuge. The columns were stored on ice with the polysome buffer in it (last wash). Once the RNaseI-treatment of lysate completed, 8.7 µL of SUPERaseIn RNase inhibitor was added to the reaction, gently mixed with the help of a pipette and stored on ice. The S-400 HR columns were spun at 2000 rpm for 1 minute to remove the polysome buffer. RNaseI-treated lysate was applied on the top of the column and spun at 2400 rpm for two minutes. The flow through was transferred to a fresh Eppendorf tube and RNA was extracted using 1.0 mL TriZol reagent. The RNA contain ribosome protected fragments (RPF) of mRNA as well as ribosomal RNA. Approximately 20-25 µg of RNaseI-treated RNAs were separated on 15% UREA-PAGE to purify RPF which are in the range of 26-34 nt. Radiolabelled RNA marker of length 28 and 30 nt were used to size select the ribosome protected RNA fragment (RPF). The urea polyacrylamide gel was run at 16 W for 1 hour with the samples. Once the bromophenol blue reached the bottom of the gel, the power was switched off and the gel was taken out of the running apparatus. One glass plate was removed and the gel was wrapped in saran wrap and exposed to Storage Phosphor Screens (GE Healthcare; BAS IP MS 2025 E, Cat no. 28-9564-75) for 30 minutes, and scanned (GE Healthcare; Typhoon FLA 9500 IP, Cat no. 29-1885-90). Based on the position of markers, the gel piece corresponding to approximately 26-34 nt was excised and transfer to an RNase-free Eppendorf tube. The gel piece was ground to a fine powder and 600 µL of RNA extraction buffer (0.5 M Ammonium Acetate, 1 mM EDTA pH 8.0, and 0.1% SDS) was added and the tubes were incubated at 25°C overnight. Next day, the slurry was transferred to filter tubes (from Art-seq kit) and spun for 5 minutes at 5000 rpm. The flow-through was collected in a new RNase-free Eppendorf tube and precipitated with 1.0 µL of glycogen and 900 µL isopropanol overnight in -20°C freezer. The RNA solution was centrifuged at 13000 rpm for 20 minutes in a cold centrifuge to pellet RNA. RNA pellet was washed with 80% ethanol once, air dried and resuspended in 20 µL of RNase-free water. m6Am-Exo-Seq (Sendinc et al., 2019) is a variation of m6A-IP seq. In this protocol, first, the m7G-capped RNA fragments (~100-200 nt) from poly(A)+ RNAs are enriched by multiple enzymatic treatments followed by m6A IP-seq to get m6A modification near the 5´  end of the mRNAs.  Approximately 8.0 µg of poly(A)+ RNAs from biological triplicates from Pcif1+/- and Pcif1-/- mouse testes were fragmented with RNA fragmentation buffer (Ambion) in a total volume of 20 µL for 7.30 minutes at 70°C in a thermcycler to obtain RNA fragments of ~100-200nt length. Fragmentation reaction was stopped by transferring the RNAs quickly to ice and adding 2.2 µL 10x stop solution (Ambion). The fragmented RNAs were purified with RNA Clean & Concentrator-5 (Zymo Research, R1014) and eluted in 17 µL water. Fragmented RNAs were 5´ phosphorylated by adding 10x, 2 µL T4 DNA ligase buffer (NEB) and 20 units of T4 PNK (ThermoScientific) in a final reaction volume of 20 µL. The phosphorylation reaction was carried out for 1.30 hour at 37°C. The 5´ phosphorylated RNA fragments were degraded by Terminator? 5´-Phosphate-Dependent Exonuclease (Epibio). To the phosphorylation reaction, 14 µL of water, 4 µL 10x Exonuclease buffer A and 2 µL Terminator exonuclease was added and the reaction mixture was incubated at 30°C for 3 hours. RNAs were purified from above reaction with RNA Clean & Concentrator-5 and eluted in 15 µL water. At this point we get 300-400 ng of m7G-cap-enriched RNA fragments from each sample. The RNA obtained from above reaction were directly used for m6A-IP (Wojtas et al., 2017) with some modifications. First, 100 µL of protein A dynabeads (Life Technologies; 10002D) were washed twice with PXL buffer (1X PBS, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% NP-40) and blocked with 1 µg/µL BSA in 400 µL PXL buffer for 45 minutes. Blocked beads were then incubated with 5 µg of anti-m6A antibody (Synaptic Systems, Cat. no. 202003) in 200 µL PXL buffer supplemented with 4 µL of RNasin RNase inhibitor (Promega; N2611) for one hour at room temperature. Antibody-conjugated beads were washed twice with PXL buffer and finally resuspended in 400 µL PXL buffer plus 4 µL RNAsin RNase inhibitor. Fragmented m7G-cap enriched RNAs were added and beads were incubated at 4°C for two hours with rotation. 10% of the RNA was kept separately and used to prepare input libraries. After two hours incubation, the beads were washed twice by ice-cold Nelson low-salt buffer (15 mM Tris at pH 7.5, 5 mM EDTA), once by ice-cold Nelson high-salt buffer (15 mM Tris at pH 7.5, 5 mM EDTA, 2.5 mM EGTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 M NaCl), once by ice-cold Nelson stringent wash buffer (15 mM Tris at pH 7.5, 5 mM EDTA, 2.5 mM EGTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 120 mM NaCl, 25 mM KCl), and last by ice-cold NT-2 buffer (50 mM Tris at pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40). Antibody-bound RNAs were eluted by adding 1mL TRIzol (Invitrogen) directly to the beads. RNAs were extracted and precipitated with Glycogen and ethanol overnight in -20°C, and used directly for the library preparation. The TruSeq Stranded Total RNA kit with Ribo-Zero Gold (Illumina, Cat. No. 20020612) was used for library preparation with 400 ng of total RNA as input. Library molarity and quality was assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies). Libraries were diluted at 2 nM and pooled before the clustering process on a HiSeq 4000 Single Read flow cell. Paired-end reads of 100 bases were generated using the TruSeq SBS reagents on the Illumina HiSeq 4000 sequencer (iGE3 Genomics Platform, University of Geneva). For ribosomal profiling the ribosomal RNAs were depleted from both input RNAs (400 ng) and RPF RNAs with mouse ribosomal RNAs probe from siTOOLs (mouse riboPOOLs) following their protocol. The ribosomal RNA-depleted input and RPF RNAs were used to make next generation sequencing library with TruSeq Ribo Profile (mammalian) kit from illumina. The libraries were sequenced at Genomics platform (University of Geneva). The m6Am-Exo-Seq libraries were prepared with NEBNext® Ultra? II Directional RNA Library Prep Kit for Illumina® (E7765) according to the protocol 3 of the kit (Protocol for use with FFPE RNA, step 3.5 onwards). The final PCR was performed for 15 cycles (98°C ? 30 s, 12 cycle of 98°C ? 10 s, 65°C ? 75 s and final extension at 65°C ? 5 minutes). The libraries were sequenced on HiSeq4000 machine at genomics platform (University of Geneva)"
GEO Accession
GSM4568887