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===IVT Protocol=== *If need to make more transposome, do first 2 steps. If not goto step 3. '''Generation of transposomes''' 1. annealing of ME sequence to T7 transposon sequence a. Make 100 uM stock solution of tranposon and ME only bottom end. b. Incubate 15 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 2 minutes, then cool to 14C at 0.1 C/s. c. Oligo's now at 30 uM in 50 uL. 2. transposome complex generation, run controls!!! *add the below components into one tube and incubate for 20 minutes at RT a. Add 0.5 uL of 100% sterile glycerol to tube b. Add 0.5 uL of annealed transposon to the 100% sterile glycerol and mix well c. Add 1.0 uL of transposase to well. *store at -20, is good for a year 3. Prepare samples, lyse cells with lysis buffer CELL WASHING - (wash cells with PBS) a. count cells, spin down all cells at 250Xg for 5 minutes b. wash with 1X PBS, centrifuge 250Xg for 5 minutes, resuspend to 1000 cells/uL c. Count cells again to be sure of cells/uL amount. CELL LYSIS a. dilute Lysis Buffer (LB) to 2X, or as needed (from 10X stock) b. add 1 uL LB to sample tubes c. add 1 uL cells to sample tubes with LB in it, mix briefly (mineral oil optional) d. transposition ready LYSIS BUFFER NOTES *Lysis buffer is: 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. 4. transposition reaction. Add all components and incubate at 37C FOR 30 MINUTES 1.0 uL 5X Custom Tagmentation buffer 2.0 uL lysed cells/pure genomic DNA 1.0 uL T7 transposomes (5X tn5-059, T7-nXTv2_i5-1) 1.0 uL N-H2O ___________ 5.0 uL total solution 5. Bring to 15 uL with N-H2O. Add 15 uL 8M guanidine HCl for a total of 30 uL. **For SPRI beads add 1.8X (54 uL) beads per sample. and follow beads protocol **'''ELUTE IN 9.6, leave beads in solution''' 6. Fill in reaction *Add 2.4 uL of 5X taq polymerase, mix thoroughly. Run at 72C for 3 minutes. 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!!!! 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 _______________________ 20 uL total b. Incubate reactions at 37C overnight for ~16-19 hours. (>10 uM limiting nucleotide) 8. Clean with Zymo RNA clean and concentrator, put solution and all beads on column. *elute samples in 10 uL of N-H2O *quanitate with Qubit or on TBU gel. '''AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO''' sample list 21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1 22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2 23. i503_shrt-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 24. i503_shrt-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 25. i505_shrt-i701_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 26. i505_shrt-i702_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 27. i506_long-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 28. i506_long-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 29. i502_long-i703_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 30. i502_long-i704_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 31. i503_shrt-i703_long, 8/27/2015, 4. T7-nXTv2_i5-1, 5X tn5-059, NTC *Amounts of RNA and water to add for MMLV RT (50 ng RNA) 9. 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 a. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 11.5 uL with Nuclease free H2O b. heat the mixture to 70C fo 3 minutes. Immediately cool on ice. c. Add the following to the reaction. 4 uL 5X first strand buffer 2 uL dNTP mix 2 uL 100 mM DTT .5 uL SMART MMLV RT and mix (ADD LAST!!!!!) ___________________________ 20 uL total d. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes. e. Terminate the reaction by heating at 70C for 10 minutes 10. RNase H digestion *Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction. a. Add 1 uL of 10X diluted Rnase H to the reaction. b. Incubate for 20 minutes at 37C. 11. Second strand synthesis: Adding P-sss_nXTv2_prmr primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since P-sss_nXTv2_prmr has a high Tm. a. Add 2.5 uL of 20 uM P-sss_nXTv2_prmr to each reaction. b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice. c. Add 5.875 uL taq5X to reactions, Incubate at 72C for 8 minutes 12. Clean DNA with AMPURE beads depending. elute in 20 uL and concentreate. *'''USED AMPURE DNA BEADS AT 1:1.8X RATIO''' *add 45 uL N-H2O *add 135 uL beads per reaction *elute on 20 uL 13. After elution in 20 uL, use vaccum centrifuge commentator to concentrate sample size to ~4-5 uL per sample. *Also ~4-5 uL per sample is preferred for transposition, which has always been performed in low volume solutions. '''RAN FOR 16 MINUTES''' 14. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6 1 uL custom tagmentation buffer X uL cDNA sample X uL Nuclase free H2O 1 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST) ___________ 5 uL total solution Incubate at 55C for 6 minutes, cool briefly on ice after 15. 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 16. Fill in reaction *Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. 17. PCR addition of barcodes '''USING NEXTERA XT V2 i5 AND i7 SEQUENCES!!!!!''' Samples: samples processed during [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-9-8 RNA processing for rest ofjohn chang T cell samples, first cell division, day7 Tsle and Tmp, day 90 memory, AND nXTv2 THS-seq update RNA processing] 21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1 22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2 23. i503_shrt-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 24. i503_shrt-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 25. i505_shrt-i701_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 26. i505_shrt-i702_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 27. i506_long-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 28. i506_long-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 29. i502_long-i703_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 30. i502_long-i704_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 31. i503_shrt-i703_long, 8/27/2015, 4. T7-nXTv2_i5-1, 5X tn5-059, NTC KAPA SYBR FAST qPCR mix until saturation, X35 cycles 9 uL Taq2X 2 uL nXTv2_i5 index primer, 2 uL F, (i5XX_T7-1_shrt, OR long) '''USED XXX''' 2 uL nXTv2_i7 index primer, 2 uL R, (i7XX_long or i7XX_orig) 4 uL H2O 1 uL 25X SYBR green 12 uL DNA template (use half RT reaction) _____________ 30 uL KAPA SYBR cycles: 72C 3m, 95C 30s, (95C for 10s, 59C for 30s, 72C for 1:30 min) X15, 72C for 3 min, 4C forever 55C for comp ends for taq2X '''HIGH BACKGROUND WITH 59C, NEXT TIME RUN AT 63C''' *terminate before curves saturate (usually cycle 6-8) 18. Gel Size selection *gel size select from 220-1000 bp, follow gel size selection protocol *do not need to include controls.
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