title: Odom_tRNA_evolution
design: Odom_tRNA_evolution
library_construction_protocol: Step1 RNA extraction 1. Excise 25 mg liver tissue sample (on dry ice) into Precellys tubes CK28-R, bertin technology  2. Remove the sample tube from the dry ice and immediately pipet 700 ul QIAzol Lysis Reagent into each sample tube. 3. Homogenize immediately on the TissueLyser  for 2x10" 5000 with 10" break 4. Place the sample tube containing the homogenate on the benchtop at room temperature (15-25C) for 5 min. This step promotes dissociation of nucleoprotein complexes. 5. Add 140 ul chloroform and shake it vigorously for 15s. Thorough mixing is important for subsequent phase separation. 6. Place the sample tube on the benchtop at room temperature for 2-3 min. 7. Centrifuge at >6000 x g (max. 9000 x g for Precellys tubes) for 15 min at 4C. After centrifugation, the sample separates into 3 phases: an upper, colorless, aqueous phase containing RNA; a white interphase enriched with DNA; and a lower, red, organic phase containing proteins. For tissues with an especially high fat content, an additional, clear phase may be visible below the red, organic phase. The volume of the aqueous phase should be approximately 350 ul. 8. Transfer the upper aqueous phase to a new reaction tube. Centrifuge again if a little bit of the red organic phase was transferred accidentally. This is important because it will interfere with the downstream precipitation.  9. Add 1 Vol% of isopropanol and mix thoroughly. Incubate 15 min at RT 10.  Centrifuge full-speed, 20 min, 4C. 11. Wash with 750 ul 75% EtOH 12. Centrifuge full-speed, 10 min, 4C 13. Allow pellet to air-dry 14. Dissolve pellet in 20-50 ul RNase-free water  Step2 , Turbo Dnase treat (Ambion AM1907)  10ug Total RNA, 1/10th volume DNase buffer (10ul), Turbo DNase enzyme (2ul), Water (to 100ul)  Incubate 37C 30 minutes (mix inactivation agent) 0.15 volume inactivation agent (15ul)   Step4, Ribominus treat (Invitrogen K1550-02)  Hybridization Step Instructions are provided below to perform hybridization for 1-10ug of your total RNA sample with the RiboMinus(tm) Eukaryote Probe. To process >10 ug total RNA sample, divide your sample into two samples, each containing <10 ug total RNA. 1. Set a water bath or heat block to 70-75C. 2. To a sterile, RNase-free 1.5 mL microcentrifuge tube, add the following: Total RNA (1-10 ug): <10 uL, RiboMinus(tm) Probe (15 pmol/uL): 10uL, Hybridization Buffer: 100 uL. 3. Incubate the tube at 70-75C for 5 minutes to denature RNA. 4. Allow the sample to cool to 37C slowly over a period of 30 minutes by placing the tube in a 37C water bath. To promote sequence-specific hybridization, it is important to allow slow cooling. Do not cool samples quickly by placing tubes in cold water. 5. While the sample is cooling down, proceed to Preparing Beads. 1. Resuspend RiboMinus(tm) Magnetic Beads in its bottle by thorough vortexing. 2. Pipet 750 uL of the bead suspension into a sterile, RNase-free, 1.5 mL microcentrifuge tube. 3. Place the tube with the bead suspension on a magnetic separator for 1 minute. The beads settle to the tube side that faces the magnet. Gently aspirate and discard the supernatant. 4. Add 750 uL sterile, DEPC Water to the beads and resuspend beads by slow vortexing. 5. Place tube on a magnetic separator for 1 minute. Aspirate and discard the supernatant. 6. Repeat Steps 4-5 once. 7. Resuspend beads in 750 uL Hybridization Buffer and transfer 250 uL beads to a new tube and maintain the tube at 37C for use at a later step. 8. Place the tube with 500 uL beads on a magnetic separator for 1 minute. Aspirate and discard the supernatant. 9. Resuspend beads in 200 uL Hybridization Buffer and keep the beads at 37C until use.  Removing rRNA 1. After the incubation at 37C for 30 minutes of the hybridized sample (above), briefly centrifuge the tube to collect the sample to the bottom of the tube. 2. Transfer the sample (~120 uL) to the prepared RiboMinus(tm) Magnetic beads from Step 9 (Preparing Beads, above). Mix well by pipetting up and down or low speed vortexing. 3. Incubate the tube at 37C for 15 minutes. During incubation, gently mix the contents occasionally. Briefly centrifuge the tube to collect the sample to the bottom of the tube. 4. Place the tube on a magnetic separator for 1 minute to pellet the rRNA-probe complex. Do not discard the supernatant. The supernatant contains RiboMinus(tm) RNA. 5. Place the tube with 250 uL beads from Step 7 (Preparing Beads, above) on a magnetic separator for 1 minute. Aspirate and discard the supernatant. 6. To this tube of beads, add ~320 uL supernatant containing RiboMinus(tm) RNA from Step 4, above. Mix well by pipetting up and down or low speed vortexing. 7. Incubate the tube at 37C for 15 minutes. During incubation, gently mix the contents occasionally. Briefly centrifuge the tube to collect the sample to the bottom of the tube. 8. Place the tube on a magnetic separator for 1 minute to pellet the rRNA-probe complex. Do not discard the supernatant as the supernatant contains RiboMinus(tm) RNA.  Concentrating RiboMinus(tm) RNA Using Ethanol Precipitation To ensure recovery of smaller (<200 nt) RNA, concentrate the RiboMinus(tm) RNA using ethanol precipitation as described below or using a modified RiboMinus(tm) Concentration Module (Cat. no. K1550-05) protocol with 60% ethanol (protocol included with the kit). 1. Transfer the RiboMinus(tm) RNA sample into a clean, RNAse-free 1.5 mL or 2 mL microcentrifuge tube. 2. Add the following components to RiboMinus(tm) RNA: - 1 mL glycogen (20 mg/mL) - 1/10th sample (eluted RNA) volume (30 uL for this protocol) of 3 M sodium acetate - 2.5X sample volumes (750 uL for this protocol) of 100% ethanol  Step5, Fractionate RNA (Ambion AM8740) Catalog #: AM8740 Ambion For fragmentation of 2-20 ug of RNA, the recommended reaction volume is 10 uL. 1. Bring the RNA sample volume to 9 uL with Nuclease-free Water (Ambion Cat #AM9938). 2. Add 1 uL of the 10X Fragmentation Buffer to the RNA sample. 3. Mix, spin briefly,and incubate at 70C for 15 min in a heating block, thermocycler, or water bath. 4. Add 1 uL of the Stop Solution, place it on ice until use, or store it at -80C.  Step6, PolyA tail (Ambion AM1350) Amount  Component 20 uL         RNA 36 uL       Nuclease-free Water 20 uL       5X E-PAP Buffer 10 uL       25 mM MnCl2 10 uL       10 mM ATP 4 uL      E-PAP 37C 1 Hour  Step7, Phenol clean up Sigma P1944 Add 200ul Nuclease free water to reaction Add 200ul Acidic Phenol Chloroform, mix Spin 14,000g 15 minutes Take top aqueous layer add to 3x volumes 100% Ethanol Spin 4C 14,000g 15 minutes Wash pellet 70% Ethanol ( Nuclease free water ) 4C 14,000g 5 minutes Air dry 5 minutes Dissolve 3.5ul Water  Step8, Smarter PCR cDNA synthesis kit Cat. Nos. 634925 1-3.5 ul RNA (1 ng-1 ug of poly A+ RNA or 2 ng-1 ug total RNA)* 1 ul 3' SMART CDS Primer II A (12 uM) x ul Deionized H2O 4.5 ul Total Volume Incubate the tubes at 72C in a hot-lid thermal cycler for 3 min, then reduce the temperature to 42C for 2 min.  Prepare a Master Mix for all reaction tubes at room temperature by combining the following reagents in the order shown: 2 ul 5X First-Strand Buffer 0.25 ul DTT (100 mM) 1 ul dNTP Mix (10 mM ) 1 ul SMARTer II A Oligonucleotide (12 uM) 0.25 ul RNase Inhibitor 1 ul SMARTScribe(tm) Reverse Transcriptase (100 U)* 5.5 ul Total Volume added per reaction  Incubate the tubes at 42C for 1 hour. Terminate the reaction by heating the tubes at 70C for 10 min.  Add 40 ul of TE buffer if you used total RNA as the starting material.  Prepare a PCR Master Mix for all reactions, plus one additional reaction. Combine the following reagents in the order shown: 74 ul Deionized H2O 10 ul 10X Advantage 2 PCR Buffer 2 ul 50X dNTP Mix (10 mM) 2 ul 5' PCR Primer II A (12 uM) 2 ul 50X Advantage 2 Polymerase Mix 90 ul Total Volume per reaction Place reaction in Thermal cycler 95C 2 minutes, 94C 15sec, 65C 30sec, 68C 30sec, 20 cycles, 68C 5 minutes, DNA purification column elute in 30ul  Step8 RSA I digest NEB Cat no. R0167R0167  30ul PCR 4ul NEB Buffer 4 5ul Water 1ul Enzyme Incubate 37C 4 hours Stop reaction 68C 20 minutes.  Run product on 2% agarose gel should see a smeared product >200bp.
attributes.tag: Experimental Factor: SPECIES
attributes.value: Mus musculus
attributes.tag: Experimental Factor: TISSUE
attributes.value: liver
attributes.tag: Experimental Factor: ChIP
attributes.value: not applicable(RNA-seq)