| Construction Protocol | Healthy mouse liver were isolated from Mus musculus domesticus C57BL/6J (two to three males, 10 to 12 weeks old, obtained from Charles River). The investigation was approved by the ethics committee and followed the Cambridge Institute guidelines for the use of animals in experimental studies under UK Home Office license PPL 70/7535. Healthy human livers (two to four males, unknown age) were obtained from the Addenbrookes Hospital at the University of Cambridge under license number 08-H0308-117 Liver specific transcriptional regulation. liver tissue was post-mortem fresh-frozen in liquid nitrogen. C57BL/6J mice were bred and housed under UK Home Office PPL 70/7535 licensing husbandry standards. 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. 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 treatment (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 Ribosomal RNA of 5 ug of DNase-treated total RNA was removed using the RiboZero (Epicentre RZC110424) kit following manufacturer's instructions. First Strand cDNA synthesis was performed using the Invitrogen it (11917-010) following manufacturer's instructions. Second strand cDNA synthesis was done by using a 10mM dUTP mix (Promega U1335) (dATP, dCTP, dGTP, dUTP). Make up reaction on ice Volume cDNA from first Strand cDNA synthesis 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 H 1ul, Water 56ul, Incubate 16C for 2 hours, Add 2ul T4 DNA polymerase 5 minutes 16C. Qiagen column purification column Elute 50ul water or EB. Library was prepared following Illumina TruSeq RNA sample prep Low Throughput kit instructions with the exception to treat sample with 1ul Uracil N-glycosylase (UNG) 37C 15minutes (Applied Biosystems) before PCR amplification. |
