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===Experiments needed to be done for proof of concept/procedures=== *procotols from shendure paper, [http://genome.cshlp.org/content/early/2012/03/30/gr.136242.111?top=1 shendure paper transposition] 1. annealing of ME sequence to T7 transposon sequence **a. Make 100 uM stock solution of T7tspn-top and T7tspn-bot. **b. Incubate 10 uL of each oligo (100uM) with 80 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 100 uL. **c. cool to RT at 0.1 C/s 2. transposome complex generation, run controls!!! (nextera normal, without transposon, without transposase, Jurkat DNA only, transposon only) *add the below components into one tube and incubate for 20 minutes at RT 2.5 uL of annealed transposon 2.5 uL of 100% sterile glycerol 5.0 uL of Ez-TN5 transposase *store at -20, is good for a year 3. transposition reaction *add the below into one tube and incubate for 8 minutes at 55C. 1 uL nextera HMW buffer 1 uL genomic DNA at prepared quantities (1 ng) 2 uL Nuclease free water 1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) ___________ 5 uL total solution method used in shendure paper: 4 uL nextera HMW buffer X uL genomic DNA at prepared quantities X uL Nuclease free water ______ 17.5 uL total solution add 2.5 uL of prepared transposomes 4. Protease digestion of transposase, protease inactivation To each tube, add: 1 uL Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final []) Incubate: 50 C 10 minutes, 70 C 20 minutes 5. Fill in reaction *Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera) *IVT on filled in reaction and not filled in reaction in to show fill-in is working. 6. should now be ready for IVT. *Look at Ambion's, life tech's and NEB's T7s. for which one to buy. 7. RNA fragmentation *Magnesium fragmentation or Shearing, look at what is done in RNA fragmentation kits 8. PolyA polymerase and ATP to A-tail 3'-ends 9. addition of adaptors to 3' ends and PCR amplification 10. size selection.
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