P-GSE24301-5 - P-GSE24301-5
nucleic acid library construction protocol
RNA extraction protocol; Note: This protocol is used to prepare RNA from cell cultures that will be used for the isolation of microRNAs. Materials * 75% ethanol, made with DNase/RNase-free water. * TRIzol® reagent: Invitrogen, cat. no. 15596-026 (100 ml) or 15596-018 (200 ml). * DNase/RNase-free water: Invitrogen cat. no. 10977. * 75% ethanol, prepared with DNase/RNase-free water. Procedure 1. Pellet cells from 5-10 plates by centrifugation in 50 ml disposable polypropylene tubes (approximately 1000 x g, 5 min.). 2. Discard the supernatant. Resuspend the pellet in 5 ml Drosophila PBS; transfer to a 15 ml polypropylene tube. 3. Pellet cells again by centrifugation. 4. Remove supernatant. 5. Lyse the pellet in TRIzol reagent (0.75 ml per 5-10 x 107 cells) by repetitive pipetting. For 5 plates of cells at 5x106 cells/ml, use 2 ml TRIzol. 6. Incubate at room temperature for 5 minutes. 7. Add 0.267 ml chloroform per ml of TRIzol used in step 5. 8. Shake tubes vigorously for 15 seconds. 9. Incubate at room temperature for 2 minutes. 10. Centrifuge for 15 minutes at 4degC at 12,000 x g. 11. Transfer the top (aqueous) phases to clean tubes. 12. Precipitate the RNA from the aqueous phase by adding 0.67 ml of isopropanol per ml of TRIzol used in step 5. 13. Invert tubes once to mix gently. 14. Incubate samples at -20º overnight. 15. Centrifuge for 10 minutes at 4° at 12,000 x g. 16. Remove supernatant. 17. Wash RNA pellet once with 75% ethanol, 0.7 ml per microfuge tube. 18. Vortex briefly. 19. Centrifuge at 7,500 x g for 5 minutes at 4°. 20. Let pellet air dry for 10 minutes. Note: DO NOT let the pellet dry completely. 21. Dissolve in RNase-free water. 22. Incubate at 37° overnight to dissolve the RNA. Determine concentration by absorbance, using a Nanodrop® ND-1000 Spectrophotometer. Store at -80°.  MRNA purfication from total RNA for Illumina seqencing protocol;  Standard Illumina mRNA Purification from Total RNA 1. Dilute 10 ug Total RNA nuclease-free H2O to 50µL in a 1.5 mL RNase free non-sticky eppendorf tube (Ambion). 2. Heat at 65°C for 5 minutes to disrupt the secondary structures, and place on ice. 3. Meanwhile, aliquot 100µL of dynal oligo(dT) beads into a 1.5mL RNase free non-sticky eppendorf tube (Ambion). 4. Wash the beads twice with 100µL Binding Buffer. 5. Resuspend the beads in 50µL Binding Buffer, and add the 50µL of total RNA sample from step 1.2; rotate at RT for 5 minutes. 6. Remove the supernatant and wash the beads twice with 100µL of Washing Buffer B. 7. Aliquot 80µL of Binding Buffer to a fresh 1.5mL RNase free non-sticky eppendorf tube (Ambion). 8. Add 20µL of 10mM Tris-HCl to the beads, heat at 800C for 2 minutes to elute mRNA from the beads. Immediately put on the magnet stand and transfer the supernatant (mRNA) to the tube from step 1.7. And add 100µL of Washing Buffer B to the beads. 9. Heat the samples at 65°C for 5 minutes to disrupt the secondary structures, and place on ice. 10. Meanwhile Wash the bead twice with 100µL of Washing Buffer B. 11. Add 100µL of mRNA sample from step 9; rotate at RT for 5 minutes. 12. Remove the supernatant and wash the beads twice with 100µL of Washing Buffer B. 13. Add 10 or 12 µL of 10mM Tris-HCl to the beads, heat at 800C for 2 minutes to elute mRNA from the beads. Immediately put on the magnet stand and transfer the supernatant (mRNA) to a fresh 200µL thin wall PCR tube, and there should be ~9 or 11 µL of mRNA.  A Gel purification of RNA sample1. Spike 50 ug of total RNA with 32P-labeled 19bp and 24bp oligos (~10,000 counts per second) and load the sample in a 15% polyacrylamide/urea gel (Sequagel, National Diagnostics). Run the gel in 1x TBE at constant 10W for 1-2 hours. Expose the gel for 10 minutes in Phosphoimager.2. Excise the band corresponding to the desired size of small RNA (~19-24 nt) into Eppendorf tubes, including the radio-labeled oligos. Add 400uL of 0.4M NaCl. Freeze rapidly in dry ice. Incubate (thaw) for 16 hours at room temperature with agitation to maximize RNA elution.3. Spin down gel slice homogenate through micropore filter (Ultrafree-MC, 0.22um, Millipore) for 1 minute at room temperature. Add 20ug of glycogen and 2 volumes of 100% Ethanol (RNase-free). Incubate at -20oC for 4 hours.4. Spin the sample at 4oC for 30 minutes. Wash the pellet with 70% EtOH, air dry for 2 min and resuspend in DEPC MQ water and proceed to section B.B 3'-linker ligation and gel purification of the ligated RNA product5. Ligate 50 pmol of Modban oligo (IDT:miRNA cloning linker 1) to the 3' terminus of the purified RNA sample in 1X ATP-free T4 RNA ligase buffer (50mM Tris-HCl (pH 7.5-7.6), 10mM MgCl2, 10mM DTT, 60ug/mL BSA) and 10% DMSO, with 5-10 pmol of T4 RNA ligase 2 Rnl2(1-249) (Ho et al, 2004). Incubate at room temperature for 1 hour 30 min. 6. Load the sample on a 15% polyacrylamide/urea gel as described above and expose the gel for 30 minutes.7. Excise the band corresponding to the desired size of small RNA (~36-41 nt) into Eppendorf tubes, including the ligated radio-labeled oligos.8. Proceed again with overnight elution and precipitation as in steps 2-4.C 5'-linker ligation and gel purification of the ligated RNA product.9. Ligate 50 pmol of Solexa linker to the 5' terminus of the RNA sample in 1XT4 RNA ligase buffer (Ambion) and 10% DMSO with 5-10 Units of T4 RNA ligase (Ambion). Incubate at 37oC for 1 hour 30 min.10. Load the sample on a 15% polyacrylamide/urea gel as described above and expose the gel for 1 hour.11. Excise the band corresponding to the desired size of small RNA (~68-73 nt) into Eppendorf tubes, including the ligated radio-labeled oligos.12. Proceed again with overnight elution and precipitation as in steps 2-4.D Reverse transcription and cDNA amplification.13. Reverse-transcribe the ligated RNA product by using 21 pmol of BanOne primer and 1 ul Superscript III Reverse Transcriptase (Invitrogen), for 1 hour at 50oC in 20 ul of reaction. Then, amplify 5 ul of the cDNA product with 100 pmol of primers Sol_5_SBS3 and Sol_3_Modban, and 5 Units of Taq Polymerase (New England Biolabs): [94oC 15 sec; 54oC 30 sec; 72oC 30 sec] x 5 cycles and [94oC 15 sec; 60oC 30 sec; 72oC 30 sec] x 17 cycles.14. Clean up the PCR product with phenol:chloroform and chloroform. Precipitate the amplified cDNA with Ethanol 100%.E Digestion of the spikes and gel-purification of the amplified cDNA.15. Perform PmeI (New England Biolabs) digestion of radiolabeled oligos (spikes) for 3-4 hours at 37oC and purify the amplified cDNA in a 2% low-melt agarose gel (SeaKem).16. Excise the ~ 120 bp DNA band and proceed to hot-phenol-based gel purification. Transfer aqueous phase to a new eppendorf tube and clean up with phenol:chloroform and chloroform. Precipitate the DNA with Ethanol 100% and resuspend it in 20uL water. 17. Perform Agilent test and dilute the sample to 10nM for Solexa sequencing.Required oligosModban: AMP-5 p=5'pCTGTAGGCACCATCAATdideoxyC-3' IDT Product name: miRNA cloning linker 1 Solexa linker: 5'-rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrC rUrCrUrUrCrCrGrArUrC-3' IDT 'Custom oligo' HPLC purified BanOne primer: 5'-ATTGATGGTGCCTACAG-3' IDT 'Custom oligo' HPLC purified Sol_5_SBS3 primer: 5'-AATGATACGGCGACCACCGAACACTCTTTCCCT ACACGACG-3' IDT 'Custom oligo' Desalt Sol_3_Modban primer: 5'-CAAGCAGAAGACGGCATACGATTGATGGTGCCT ACAG-3' IDT 'Custom oligo' HPLC purified 1. cluster generation2. the length of short reads, i.e., the number of sequencing cycles, should list here. in this protocol, it is 35 cycles of sequencing.3. after each cycle, a CCD camera will scan the cells to make a image.