Odom_tRNA_evolution
Odom_tRNA_evolution
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 2x10sec 5000 with 10sec 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 >6000g (max. 9000g 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) Volume 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  Step3, polyA+ enrichment (Promega PolyATtract(tm) mRNA Isolation Systems Z5310)  Annealing of the probe Instructions are provided below to perform hybridization for 1-10ug of your total RNA sample with the Promega PolyATtract(tm) mRNA Isolation Systems III and IV. 1. To a sterile, RNase-free 1.5 mL microcentrifuge tube, bring 10 ug of total RNA to a final volume of 500 ul in RNase-free water 2. place the tube in a 65C water bath or heat block for 10 min. 3. add 3ul of the biotinylated-oligo(dT) probe and 13ul 20xSSC to the RNA. Mix gently and incubate at room temperature until completely colled. This should require about 10 min.  Washing of streptavidin-paramagnetic particles 1. resuspent the manufacturer's provided streptavidin-paramagnetic particles (1 tube per sample), capture them by placing the tube on a magnetic stand 2. remove the supernatant 3. wash the streptavidin-paramagnetic particles three times with 0.5xSSC (300ul per wash), by gently flicking the bottom of the tube until the particles are resuspended, each time capturing them using the magnetic stand and carefully removing the supernatant 4. resuspend the streptavidin-paramagnetic particles in 100ul of 0.5xSSC  Capture and washing of annealed oligo(dT)-mRNA hybrids 1. add the entire contents of the annealed oligo(dT)-mRNA hybrids (520 ul) to the washed streptavidin-paramagnetic particles 2. incubate at room temperature for 10 min, mix by gently inverting every minute 3. capture the streptavidin-paramagnetic particles using the magnetic stand and carefully remove the supernatant without disturbing the streptavidin-paramagnetic particles pellet 4. wash the streptavidin-paramagnetic particles four times with 0.1xSSC (300ul per wash) by gently flicking the bottom of the tube until the particles are resuspended, each time capturing them using the magnetic stand and carefully removing the supernatant. After the final wash, remove as much supernatant as possible.  Elution of polyA+ RNA 1. resuspend the final streptavidin-paramagnetic particles in 100ul of RNase-free water and gently mix by flicking the tube 2. magnetically capture the streptavidin-paramagnetic particles and transfer the eluated polyA+ RNA in a RNase-free tube. 3. Repeat the capturing step by resuspending the streptavidin-paramagnetic particles pellet in 150ul of RNase-free water. Repeat the capturing step, pooling the eluate with the polyA+ RNA eluted before.  Concentrating polyA+ RNA using ethanol precipitation To ensure recovery of polyA+ RNA, concentrate by using ethanol precipitation. Add the following components to polyA+ RNA: - 1 mL glycogen (20 mg/mL) - 1/10th sample (eluted RNA) volume (15 uL for this protocol) of 3 M sodium acetate - 2.5X sample volumes (375 uL for this protocol) of 100% ethanol  Step4, 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.  Concentrating fractionated polyA+ RNA using ethanol precipitation Add the following components to fractionated polyA+ RNA: - 1 mL glycogen (20 mg/mL) - 1/10th sample (eluted RNA) volume of 3 M sodium acetate - 2.5X sample volumes of 100% ethanol  Step5, Synthesize the 1st strand cDNA 1. add 1 ul of random primers to 11.1 ul of fractionated polyA+ RNA 2. place the tube in a 65C PCR thermal cycler for 5 min, and chill on ice. 3. mix the following reagents in the order listed: 5x 1st strand buffer 4ul 100 mM DTT           2ul 25 mM dNTP mix    0.4 ul RNase OUT         0.5 ul 4. add the mixture to the PCR tube and mix well 5. heat the sample at 25C for 2 min 6. add 1ul SuperScript II to the sample and incubate in a thermal cycler: a. 25C for 10 min b. 42C for 50 min c. 70C for 15 min d. hold at 4C  Step6, Synthesize the 2nd strand cDNA 1. add: 62.8ul of water to the 1st strand cDNA synthesis mix 10ul GEX second strand buffer 1.2ul 25mM dNTP mix 2. incubate on ice for 5 min 3. add 1ul of RNaseH and 5ul of DNA pol I 4. mix and incubate at 16C in a thermal cycler for 2.5h 5. follow the instructions in the QIAquick PCR Purification Kit to purify the sample and elute in 50ul of EB  Step7, A. End Repair Pipett the following mix direct into PCR tubes and keep on ice. Fractionated poly A+ RNA 50.0 ul Water  25.0 ul T4 DNA Ligase buffer with 10 mM ATP  10.0 ul dNTP mix (each 10 mM) 4.0 ul T4 DNA polymerase     5.0 ul Klenow DNA polymerase 1.0 ul T4 PNK  5.0 ul Total 100.0 ul  1. Incubate 30 min at 20C in a thermal cycler. 2. Cleanup samples using the DNA Clean&Concentrator-5 kit, (Zymo research,USA), following the manufacturer's protocol. 3. Elute with 32 ul EB preheated to 50C. Chill on ice.  Step8, Add "A" Bases Pipett the following mix direct into PCR tubes and keep on ice. DNA sample       32.0 ul Klenow buffer     5.0 ul dATP (1mM)       10.0 ul Klenow (3'-5' exo minus; 5 U/ul)  3.0 ul Total    50.0 ul  1. Incubate 30 min at 37C in a thermal cycler. 2. Cleanup samples using the DNA Clean&Concentrator-5 kit, (Zymo research,USA), following the manufacturer's protocol. 3. Elute with 8 ul EB preheated to 50C. Chill on ice.  Step9, Ligate sequencing Adapters to DNA fragments Pipett the following mix in 1.5 ml tubes. DNA sample         8.0 ul DNA Ligase buffer 12.5 ul Adapter Oligo mix (from Illumina)  1.0 ul DNA ligase         2.5 ul Total 25.0 ul  1. Incubate 15 min at RT. (Requires Rapid DNA Ligase, else incubate 16 h at 16C. 2. Cleanup samples using the DNA Clean&Concentrator-5 kit, (Zymo research,USA), following the manufacturer's protocol. 3. Elute with 23 ul EB preheated to 50C. Chill on ice.  Step10, Enrich Adapter-Modified DNA by PCR: Pipette the following mix direct into PCR tubes and keep on ice. DNA sample    23.0 ul Phusion DNA pol 2x master mix 25.0 ul PCR primer 1.0 1.0 ul PCR primer 2.0 1.0 ul Total         50.0 ul  Run the PCR using the following program: Step1:    98C    30 sec Step2:    98C    10 sec Step3:    65C    30 sec Step4:    72C    30 sec Step5 GOTO Step2 14 times Step6:    72C     5 min Step7:     4C    HOLD  Step11, purify the RNAseq library 1. prepare a 2% agarose gel in 1xTAE 2. add 8ul of 6xDNA loading dye to the PCR-enriched DNA 3. loading: use 2ul of 100bp DNA ladder (with 6xDNA loading dye) and sample 4. run the gel at 120V for 60 min 5. excise the 200 bp (+/- 25 bp) region of a gel with a scalpel or gene catcher
Experimental Factor: SPECIES
Rattus norvegicus
Experimental Factor: TISSUE
liver
Experimental Factor: ChIP
not applicable(RNA-seq)