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===IVT Protocol=== *If need to make more transposome, do first 2 steps. 1. annealing of ME sequence to T7 transposon sequence **a. Make 100 uM stock solution of T7tspn-top2 and T7tspn-bot. **b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL. **c. cool to RT at 0.1 C/s 2. transposome complex generation, run controls!!! *add the below components into one tube and incubate for 20 minutes at RT 1.25 uL of annealed transposon 1.25 uL of 100% sterile glycerol 2.50 uL of Ez-TN5 transposase *store at -20, is good for a year 3. Prepare samples, lyse cells with lysis buffer use pure GM12878 DNA Samples: 1. 1000 cells IVT 2. 500 cells IVT 3. 100 cells IVT 4. 6 ng purified DNA IVT 5. 600 pg purified DNA IVT 6. 60 pg purified DNA IVT 13. 1000 cells MEFs IVT 15. 1000 cells lysed without transposome complex IVT 16. pure DNA only (6 ng) IVT 17. Nuclease free H20 only IVT *Make 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O. *Prepare aliquots of lymphocytes GM12878 (have GM20431, GM12878 and MEFs) that contain 1000, 500, 100 cells. **spin down cells to concentrate them as necessary. *Prepare 2X LB from 10X buffer. mineral oil optional. *if needed make 4 or 6 uL solutions. keep 1:1 ratio of cells:lysis buffer *incubate below mixtures at 37C for 30 mins. {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''1. 1K IVT''' | align="center" style="background:#f0f0f0;"|'''2. 500 IVT''' | align="center" style="background:#f0f0f0;"|'''3. 100 IVT''' | align="center" style="background:#f0f0f0;"|'''7. 1K nxta''' | align="center" style="background:#f0f0f0;"|'''8. 500 nxta''' | align="center" style="background:#f0f0f0;"|'''9. 100 nxta''' | align="center" style="background:#f0f0f0;"|'''13. 1K MEF IVT''' | align="center" style="background:#f0f0f0;"|'''14. 1K MEF nxta''' | align="center" style="background:#f0f0f0;"|'''15. 1K IVT con''' | align="center" style="background:#f0f0f0;"|'''17. NTC IVT''' | align="center" style="background:#f0f0f0;"|'''18. 1K nxta con''' | align="center" style="background:#f0f0f0;"|'''20. NTC nxta''' |- | cells||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul PBS||1 ul||1 ul PBS |- | 2X LB||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul||1 ul |- | |} 4. transposition reaction, using (T7tspn-top2) *add the below into one tube and incubate for 10 minutes at 55C. 1 uL nextera LMW buffer 2 uL lysed/pure genomic DNA (X ng/pg DNA) 1.2 uL Nuclease free water .8 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL) ___________ 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 5. 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: 50C 10 minutes, 70C 20 minutes 6. Fill in reaction *Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera) 7. Maxiscript (Ambion) T7 Protocol, IVT *DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification. a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) on ice, but keep 10X transcription buffer at room temp b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!! bring to 20 uL with Nuclease free water X uL DNA template (list 1 ug) 2 uL 10X Transcription Buffer 1 uL 10 mM ATP 1 uL 10 mM CTP 1 uL 10 mM GTP 1 uL 10 mM UTP 2 uL T7 Enzyme Mix b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide) 8. Clean RNA with with Zymo RNA Clean & Concentrator-5 Kit *can quantitate with Qubit if needed 9. Perform RNase III fragmentation (NEB): Starting Material: Purified mRNA (50β250 nanograms) 1. Add 5' end blocking DNA primer and do annealing to form DNA-RNA hybrid. *a. add 2 uL of blocking oligo (10 uM) to aliquot RNA sample that will be used in step 2. use T7-frag-block-top2 for T7-top2. T7-frag-block for T7-top3 *b. Incubate at 95C for 2 minutes. *c. cool to RT at 0.1 C/s 2. Mix the following components in a sterile PCR tube: X uL Purified mRNA + blocking primer (50-250 nanograms) .5 uL RNase III (1 unit/ΞΌl) 1 uL RNase III Reaction Buffer (10X) 5.5 uL Nuclease-Free Water add in DNA primer to protect 5' end since don't want degradation?? _______ 10 uL total volume 3. Incubate in a preheated thermal cycler for 5 minutes at 37Β°C. 4. Transfer tube to ice. 10. (ONLY DID ZYMO CLEANUP, STILL WORKED FINE 2012/08/28 update) DNase I digestion and Zymo RNA clean and concentrator cleanup. removes dsDNA. does not remove ss or DNA:RNA hybrids at high efficiency, 1:500 that of dsDNA. better than nothing... *Resuspend in appropriate volume in nuclease free H2O. *not doing protease digestion since seems it wasnt as effective in previous [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-6-6 RNase III fragmentation] 11. Quanitate RNA with QUbit 12. Poly(A) Addition with polyA polymerase (Enzymatics) *enzymatics PolyA polymerase. a. assemble reaction: 2 uL 5X SMART MMLV first strand buffer (rather than 10X polyA buffer) 1 uL polyA enzyme 1 uL 10 mM ATP bring to 10 uL with RNA or w/e b. Incubate at 37C for 10 minutes c. Heat inactivate at 70C for 20 minutes. (rui and NEB) 13. single strand synthesis MMLV RT (Clontech) *Followed protocol for [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/SMART_MMLV_RT SMART MMLV Reverse Transcriptase] 20 uL reaction 1. Add 2.5 uL 20 uM primer stock to RNA sample. Bring to final volume of 12.5 uL with Nuclease free H2O (one from BENG160 class, T20VN_PE_R) 2. heat the mixture to 70C fo 3 minutes. Immediately cool on ice. 3. Add the following to the reaction. 2 uL 5X first strand buffer 2 uL dNTP mix 2 uL 100 uM DTT 1 uL N-H20 .5 uL SMART MMLV RT and mix (ADD LAST!!!!!) ____ 20 uL total 4. Incuvate at 42C for 60 minutes 5. Terminate the reaction by heating at 70C for 10 minutes 14. second strand synthesis (qPCR) (KAPA), addition of barcodes Samples: Index 49, N2 adaptor 1. 1000 cells IVT Index 50, N2 adaptor 2. 500 cells IVT Index 51, N2 adaptor 3. 100 cells IVT Index 52, N2 adaptor 4. 6 ng purified DNA IVT Index 53, N2 adaptor 5. 600 pg purified DNA IVT Index 54, N2 adaptor 6. 60 pg purified DNA IVT Index 61, N2 adaptor 13. 1000 cells MEFs IVT Index 63, N2 adaptor 15. 1000 cells lysed without transposome complex IVT Index 64, N2 adaptor 16. pure DNA only (6 ng) IVT Index 65, N2 adaptor 17. Nuclease free H20 only IVT KAPA SYBR FAST qPCR mix until saturation, X35 cycles 25 uL KAPA SYBR 4 uL primers, 2 uL F, 2 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX]) 1 uL H2O 20 uL DNA template (use whole RT reaction) KAPA SYBR cycles: 98C 3min, (98C for 30s, 60C for 30s, 72C for 2 min) X35, 72C for 5 min, 4C forever *terminate before curves saturate (usually cycle 6-7) 15.qiaquick cleanup *can quanitate with nanodrop *run qiaquick to clean sample before performing qPCR. 16. Gel Size selection *gel size select from 400-800 bp, follow gel size selection protocol *do not need to include controls. 17. Cloning and Transformation, then genewiz sequencing for verification of inserts 18. Submit for sequencing if genewiz sequencing checks out.
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