Transcription profiling by high throughput sequencing of long non-coding RNAs across multiple rodent species
Transcription profiling by high throughput sequencing of long non-coding RNAs across multiple rodent species
do933_RNAseq_liver_rno8
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 . Step  First Strand cDNA synthesis ( Invitrogen 11917-010 ). mRNA 10ul, oligo dT 1ul, random primer 1ul, Incubate 70C 5 minutes then transfer to ICE for a few minutes. 5x FSS buffer  5ul, DTT2.5ul, dNTP1ul, RNase out 1ul, Water 2.5ul. Incubate 25C for 2 minutes then:1ul Superscript 2 42C 1 hour ( minimal ) Or 1ul Superscript 3 46C 1 hour ( minimal )70C 15 minutes to inactivate. Use Qiagen PCR column to remove dNTP from FSS reaction. Elute 50ul water or EB. Step 4 Second Strand Synthesis Make up 10mM DUTP mix ( Promega U1335 )(dATP, dCTP, dGTP, dUTP) Make up reaction on ice Volume cDNA from step 3 50ul 5x FSS buffer, 4ul DTT 2ul, 5xSSS buffer 30ul DU/NTP mix 3ul, E.coli DNA ligase 1ul,  E.Coli DNA polymerase  3ul, E.Coli Rnase H1ul,  Water 56ul, Incubate 16C for 2 hours, Add 2ul T4 DNA polymerase 5 minutes 16C. Qiagen column purification column Elute 50ul water or EB. Nanodrop sample ( in my experience i had around 500ng cDNA ). Take 250ng (store any remainder -20C) and make up to 300ul with water ( arbitrary amount ). Step 5 Sonication Bioruptor 2 x 5minutes 30 sec ON 30 sec OFF, full power changing ice in between. Ethanol PPT. Prepare cDNA for sequencing by attaching adapters as per Illumina manufacturesrs instructions, exept for modification, before PCR amplification treat sample with 1ul Uracil N-glycosylase (UNG) 37C 15minutes ( Applied Biosystems ).