P-GSE24310-2 - P-GSE24310-2
nucleic acid library construction protocol
1. Freeze 3-5 days old adult flies at -80C 2. Vortex frozen flies in a 15ml conical tube. 3. Sieve heads and bodies by mesh # 20 and # 40. Bodies are recovered from #20 fraction. Legs and wings are mostly lost in this procedure. Proceed directly to total RNA isolation or freeze the samples at -80C. Samples are homogenized in 100ul Hepes-NP40 buffer using plastic pestles and then 900ul Hepes-NP40 is added .Lysates are cleared by centrifugation at 4C for 10 minutes at 10,000g. Cleared extract was split and incubated with rabbit polyclonal AGO1 antibody (1:20; lot number 113754, Abcam) for 4 h at 4 uC. AGO1 antibodies were isolated by adding protein G beads (1:10; Roche) for 1 h. Beads were washed six times each for 10 min in buffer B (30 mM HEPES, pH 7.4, 800 mM NaCl, 2 mM MgCl2, 0.1% NP-40), which contained equal supplements as buffer A. The immunoprecipitation was analysed by western blotting using anti-AGO1 (1:2,000; Abcam). AGO1-associated RNAs were isolated with phenol/chloroform and ethanol precipitated. 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 is 35 cycles of sequencing.3. after each cycle, a CCD camera will scan the cells to make a image.