Brandon:LabNotes/Project1/2015-6-18
tn5-059 NXTA_ME_B vs ez-tn5 NXTA_ME_B for secondary fragmentation troubleshooting R3[edit]
- Titration_of_tn5-059_NXTA_ME_B_for_secondary_fragmentation.2C_and_fill-in_only_during_PCR_step.3F didn't work.
- Then tn5-059 NXTA_ME_B for secondary fragmentation trial with cell samples, with ez-tn5 nxta_me_B control didn't work. Think MMLV is dead.
- new MMLV and all reagents
- new dNTPs (advantage from MMLV)
- new random hexamers
- new RNase H (enzymatics)
- new sss_NPA_prmr (6/3/2015)
- new taq5x (5/20/15)
- new T7-top2-PCR-iaf2 (4/2/2015)
- new RNA XP SPRI beads
- ez-tn5 control
- will test with 1000 cell, 6 ng DNA john chang samples to make sure they work. when preparing 100 cell, 500 cell GM12878 libraries used custom transposons with ez-tn5 for secondary fragmentation and adaptor addition. tn5059 should be better.
- using RNA from: Naive and activated T cells from john chang Lab, pilot with Tn5-059 100 cell samples, mouse, Trial 2 since it is not the experimental samples
AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO
- sample list
1. idx49, 2015-5-26, 9. t7-top2, 5X tn5-059, 1000 cells, N1, ez-tn5 2. idx50, 2015-5-26, 9. t7-top2, 5X tn5-059, 1000 cells, N1, tn5-059 3. idx51, 2015-5-26, 11. t7-top2, 5X tn5-059, 1000 cells, A1, ez-tn5 4. idx52, 2015-5-26, 11. t7-top2, 5X tn5-059, 1000 cells, A1, tn5-059 5. idx53, 2015-5-26, 13. t7-top2, 5X tn5-059, 6 ng pure DNA, ez-tn5 6. idx54, 2015-5-26, 13. t7-top2, 5X tn5-059, 6 ng pure DNA, tn5-059 7. idx55, 2015-5-26, 14 t7-top2, 5X tn5-059, NTC, ez-tn5 8. idx56, 2015-5-26, 14 t7-top2, 5X tn5-059, NTC, tn5-059
- Amounts of RNA and water to add for MMLV RT (50 ng RNA)
' | ul for 50 ng | ' | N-H20 |
idx49, 2015-5-26, 9. t7-top2, 5X tn5-059, 1000 cells, N1, ez-tn5 | 0.926128791 | 8.073871209 | |
idx50, 2015-5-26, 9. t7-top2, 5X tn5-059, 1000 cells, N1, tn5-059 | 0.926128791 | 8.073871209 | |
idx51, 2015-5-26, 11. t7-top2, 5X tn5-059, 1000 cells, A1, ez-tn5 | 0.764755128 | 8.235244872 | |
idx52, 2015-5-26, 11. t7-top2, 5X tn5-059, 1000 cells, A1, tn5-059 | 0.764755128 | 8.235244872 | |
idx53, 2015-5-26, 13. t7-top2, 5X tn5-059, 6 ng pure DNA, ez-tn5 | 0.724059485 | /8 | 8.275940515 |
idx54, 2015-5-26, 13. t7-top2, 5X tn5-059, 6 ng pure DNA, tn5-059 | 0.724059485 | /8 | 8.275940515 |
idx55, 2015-5-26, 14 t7-top2, 5X tn5-059, NTC, | 1 | 8 | |
idx56, 2015-5-26, 14 t7-top2, 5X tn5-059, NTC, | 1 | 8 | |
8. single strand synthesis MMLV RT (Clontech)
- Followed protocol for SMART MMLV Reverse Transcriptase
20 uL reaction 1. 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 2. heat the mixture to 70C fo 3 minutes. Immediately cool on ice. 3. 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 4. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes. 5. Terminate the reaction by heating at 70C for 10 minutes
9. 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.
10. Second strand synthesis: Adding sss_NPA_prmr primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_NPA_prmr has a high Tm.
a. Add 2.5 uL of 20 uM sss_NPA_prmr to each reaction. b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice. d. Add polymerases etc to reactions taq5X: Add 5.875 uL, Incubate at 72C for 8 minutes
11. Clean DNA with AMPURE beads depending. elute in 20 uL and concentreate. (can also use qiaquick)
- USED AMPURE DNA BEADS AT 1:1.8X RATIO
- add 45 uL N-H2O
- add 135 uL beads per reaction
- elute on 20 uL
can quantitate with Qubit or on TBU gel.
11.5. Eluted DNA into 20 uL per sample, since when eluting in smaller amounts more sample is lost. Thus after elution used 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. Additionally less transposome will be needed when doing that.
RAN FOR 16 MINUTES
12. Fragmenting and 3' End tagging with Custom Nextera transposome
USING NXTA_ME_B WITH TN5059, SAMPLES 2,4,6,8
EZ-TN5 NXTA_ME_B FOR CONTROL, SAMPLES 1,3,5,7
1 uL custom buffer D6 2 uL lysed/pure genomic DNA (X ng/pg DNA) X uL Nuclease free H2O 1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) ___________ 5 uL total solution Incubate at 55C for 6 minutes, cool briefly on ice after
13. 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
14. Fill in reaction
- Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
15. PCR addition of barcodes MAKE SURE TO USE NEXTERA INDEXES!!!!!
KAPA SYBR FAST qPCR mix until saturation, X35 cycles sample list 1. idx49, 2015-5-26, 9. t7-top2, 5X tn5-059, 1000 cells, N1, ez-tn5 2. idx50, 2015-5-26, 9. t7-top2, 5X tn5-059, 1000 cells, N1, tn5-059 3. idx51, 2015-5-26, 11. t7-top2, 5X tn5-059, 1000 cells, A1, ez-tn5 4. idx52, 2015-5-26, 11. t7-top2, 5X tn5-059, 1000 cells, A1, tn5-059 5. idx53, 2015-5-26, 13. t7-top2, 5X tn5-059, 6 ng pure DNA, ez-tn5 6. idx54, 2015-5-26, 13. t7-top2, 5X tn5-059, 6 ng pure DNA, tn5-059 7. idx55, 2015-5-26, 14 t7-top2, 5X tn5-059, NTC, ez-tn5 8. idx56, 2015-5-26, 14 t7-top2, 5X tn5-059, NTC, tn5-059
9 uL Taq2X 2 uL primers, 2 uL F, (T7-top2-PCR-iaf, OR iaf2, iaf3) USED iaf2 2 uL NXTA indexes PCR_R.NXTAInd[XX], SPECIFIC TO EACH SAMPLE 4 uL H2O 1 uL 25X SYBR green 12 uL DNA template _____________ 30 uL KAPA SYBR cycles: 72C 3 m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 1 min) X15, 72C for 3 min, 4C forever
- terminate before curves saturate (usually cycle 6-8)
16. Gel Size selection
- gel size select from 400-800 bp, follow gel size selection protocol
- do not need to include controls.
Results[edit]
- PCR addition of barcodes
File:2015-06-19 nxta me b troubleshooting, all new reagents, eztn5, 059.bmp
- gel after PCR addition of barcodes
File:ZhangLab 2 2015-06-19 15hr 54min-labeled.jpg
- amounts for gel size selection
' | ' | ' | ' | ' | ' |
6/19/2015 gel size selection, nxta_me_b trial R3 | 6371.31019 | 5 | |||
3821.870114 | 2.5 | for 50 ng | |||
1. idx49, 2015-5-26, 9. t7-top2, 5X tn5-059, 1000 cells, N1, ez-tn5 | 7172.110214 | 5.785270491 | 12.09969354 | ||
2. idx50, 2015-5-26, 9. t7-top2, 5X tn5-059, 1000 cells, N1, tn5-059 | 19419.83058 | 17.79547665 | 3.933583874 | ||
3. idx51, 2015-5-26, 11. t7-top2, 5X tn5-059, 1000 cells, A1, ez-tn5 | 6699.1202 | 5.321452947 | 13.15430216 | 28 | |
4. idx52, 2015-5-26, 11. t7-top2, 5X tn5-059, 1000 cells, A1, tn5-059 | 19638.61059 | 18.01001396 | 3.886726581 | ||
5. idx53, 2015-5-26, 13. t7-top2, 5X tn5-059, 6 ng pure DNA, ez-tn5 | 13045.32039 | 11.5445843 | 6.063449162 | 1.212689832 | |
6. idx54, 2015-5-26, 13. t7-top2, 5X tn5-059, 6 ng pure DNA, tn5-059 | 22875.96068 | 21.18458367 | 3.304289623 | 0.660857925 | |
7. idx55, 2015-5-26, 14 t7-top2, 5X tn5-059, NTC, ez-tn5 | 937.5000279 | -0.328434872 | 1 | ||
8. idx56, 2015-5-26, 14 t7-top2, 5X tn5-059, NTC, tn5-059 | 817.7900244 | -0.445823397 | 1 | ||
ttl | dye | ||||
36.94785392 | 7.389570783 | ||||
- gel size selection
File:ZhangLab 2 2015-06-19 18hr 01min-labeled.jpg
- gel size selection validation
File:ZhangLab 2 2015-06-19 20hr 44min-labeled.jpg
future[edit]
- balh