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===Nextera Tagmentation Protocol=== 1. See step 3 in IVT protocol. Do the cell lysis section for the "nextera tagmentation samples", samples 7,8,9,14,18. 2. Perform tagmentation on lysed cells and pure DNA. with 1:10 diluted nextera enzyme. (1:50 diluted in reaction) Dilute the enzyme: 1:10 For each rxn, used mix of: 1ul 5x LMW Buffer 2ul cell lysate 1ul diluted enzyme 1ul H2O ---------------------------- 5ul total / reaction 55C 10 min 3. Protease digestion of transposition reactions. 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: 50C 10 minutes, 70C 20 minutes 4. First step of 2-step PCR thermocycling, terminate curves before saturation. 6 ul Tagmentation reaction 25 ul KAPA SYBR FAST qPCR mix 1 ul Orange Primer (10uM) 1 ul Blue Primer (10uM) 0.5 ul Bst Pol (5U/ul) 16.5 ul H2O ---------------- 50 uL total Orange primer CCTTGCCAGCCCGCTCAG 18nt Blue primer CCTCCCTCGCGCCATCAG 18nt PCR cycling. 72C for 3 minutes is for gap fill in. 72C 3min -> 95C 30 sec -> (95C 10sec -> 58C 30 sec -> 72 3min) x 25 -> 72C 3min. Monitor the reactions on a real-time thermal cycler and terminate them before the curves reach saturation. 5. Begin 2-Step AMPure beads purification to remove primers, add index primers, for low input. (with forward read primer and reverse index primers) a. see 2-step [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/AMPure_beads_purification2#AMPure_beads_purification_protocol_2-step_.28for_.3C.3D_5_ng.29 AMpure beads protocol], start from beginning for first purification b. After cleaning, second PCR reaction to add index primers Barcodes to use: Index 55, Nxtra atpr 7. 1000 cells nextera tagmentation Index 56, Nxtra atpr 8. 500 cells nextera tagmentation Index 57, Nxtra atpr 9. 100 cells nextera tagmentation Index 58, Nxtra atpr 10. 6 ng purified DNA nextera tagmentation Index 59, Nxtra atpr 11. 600 pg purified DNA nextera tagmentation Index 60, Nxtra atpr 12. 60 pg purified DNA nextera tagmentation Index 62, Nxtra atpr 14. 1000 cells MEFs nextera tagmentation Index 66, Nxtra atpr 18. 1000 cells lysed without transposome complex nextera tagmentation Index 67, Nxtra atpr 19. pure DNA only (6 ng) nextera tagmentation Index 68, Nxtra atpr 20. Nuclease free H20 only nextera tagmentation add the following to above dry tube: 25 uL KAPA HF mix (used KAPA SYBR FAST qPCR mix instead) 1 uL adaptor1 1 uL of adaptor2 (barcode) 23 uL nuclease free H2O c. PCR cycling for addition of index primer 5 cycles 72C 3min -> 95C 30 sec -> (95C 10sec -> 60C 30 sec -> 72 1 min) x 5 -> 72C 3min. d. second phase of AMpure beads 2-step beads purification. *can run on gel and check smears to see for library. *should be ready for sequencing now?
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