Brandon:LabNotes/Project1/2015-9-8
RNA processing for rest of john chang T cell samples, first cell division, day7 Tsle and Tmp, day 90 memory, AND nXTv2 THS-seq update RNA processing[edit]
- Update of THS-seq protocol sequences to use Nextera XT V2 sequences. See that page for RNA processing instructions, which use different primers etc
- run worked on HiSeq, will run rest of samples
8/13/2015 previous RNA processing, naive t cells, day 3 T cells
AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO
- sample picking and calculations
File:T cells nXTv2 validation sample picking and planning.xlsx
- sample list
1. Idx49, 6/22/2015, 1. t7-top2, 5X tn5-059, 150 cells, mouse 1, T memory precoursor (Tmp) (CD25low CD62Lhi) 2. Idx50, 6/22/2015, 3. t7-top2, 5X tn5-059, 150 cells, mouse 1, T short lived effector (Tsle) (CD25hi CD62Llow) 3. Idx51, 6/22/2015, 5. t7-top2, 5X tn5-059, 150 cells, mouse 1, activated parental cells (CD44hi) 4. Idx52, 6/22/2015, 7. t7-top2, 5X tn5-059, 150 cells, mouse 2, T memory precoursor (Tmp) (CD25low CD62Lhi) 5. Idx53, 6/22/2015, 10. t7-top2, 5X tn5-059, 150 cells, mouse 2, T short lived effector (Tsle) (CD25hi CD62Llow) 6. Idx54, 6/22/2015, 14. t7-top2, 5X tn5-059, 150 cells, mouse 2, activated parental cells (CD44hi) 7. Idx55, 6/9/2015, 16. t7-top2, 5X tn5-059, 6 ng pure DNA 8. Idx56, 6/9/2015, 17. t7-top2, 5X tn5-059, NTC 9. Idx57, 6/9/2015, 1. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle 10. Idx58, 6/9/2015, 2. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle 11. Idx59, 6/9/2015, 3. t7-top2, 5X tn5-059, 1000 cells, day 7 Tsle 12. Idx60, 6/9/2015, 6. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp 13. Idx61,6/9/2015, 7. t7-top2, 5X tn5-059, 250 cells, day 7 Tmp 14. Idx62, 6/9/2015, 8. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp 15. Idx63, 6/17/2015, 1. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory 16. Idx64, 6/17/2015, 2. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory 17. Idx65, 6/17/2015, 3. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T central memory 18. Idx66, 6/17/2015, 7. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory 19. Idx67, 6/17/2015, 8. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory 20. Idx68, 6/17/2015, 9. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T effector memory 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)
' | 9/8/2015 RNA processing | ' | ' | ' |
uL 50 ng | H2O | |||
1 | 1. Idx49, 6/22/2015, 1. t7-top2, 5X tn5-059, 150 cells, mouse 1, T memory precoursor (Tmp) (CD25low CD62Lhi) | 4.029963775 | 4.970036225 | |
3 | 2. Idx50, 6/22/2015, 3. t7-top2, 5X tn5-059, 150 cells, mouse 1, T short lived effector (Tsle) (CD25hi CD62Llow) | 4.021654718 | 4.978345282 | |
5 | 3. Idx51, 6/22/2015, 5. t7-top2, 5X tn5-059, 150 cells, mouse 1, activated parental cells (CD44hi) | 5.170801447 | 3.829198553 | |
7 | 4. Idx52, 6/22/2015, 7. t7-top2, 5X tn5-059, 150 cells, mouse 2, T memory precoursor (Tmp) (CD25low CD62Lhi) | 6.970010025 | 2.029989975 | |
10 | 5. Idx53, 6/22/2015, 10. t7-top2, 5X tn5-059, 150 cells, mouse 2, T short lived effector (Tsle) (CD25hi CD62Llow) | 3.461639409 | 5.538360591 | |
14 | 6. Idx54, 6/22/2015, 14. t7-top2, 5X tn5-059, 150 cells, mouse 2, activated parental cells (CD44hi) | 3.027932364 | 5.972067636 | |
16 | 7. Idx55, 6/9/2015, 16. t7-top2, 5X tn5-059, 6 ng pure DNA | 1.276475454 | 7.723524546 | /8 |
17 | 8. Idx56, 6/9/2015, 17. t7-top2, 5X tn5-059, NTC | 1 | 8 | |
23 | 9. Idx57, 6/9/2015, 1. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle | 10 | -1 | |
24 | 10. Idx58, 6/9/2015, 2. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle | 10 | -1 | |
25 | 11. Idx59, 6/9/2015, 3. t7-top2, 5X tn5-059, 1000 cells, day 7 Tsle | 1.104233808 | 7.895766192 | |
28 | 12. Idx60, 6/9/2015, 6. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp | 10 | -1 | |
29 | 13. Idx61,6/9/2015, 7. t7-top2, 5X tn5-059, 250 cells, day 7 Tmp | 5.207456887 | 3.792543113 | |
30 | 14. Idx62, 6/9/2015, 8. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp | 6.48327683 | 2.51672317 | |
37 | 15. Idx63, 6/17/2015, 1. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory | 3.656925406 | 5.343074594 | |
38 | 16. Idx64, 6/17/2015, 2. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory | 4.864377142 | 4.135622858 | |
39 | 17. Idx65, 6/17/2015, 3. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T central memory | 0.975440928 | 8.024559072 | |
43 | 18. Idx66, 6/17/2015, 7. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory | 4.446580533 | 4.553419467 | |
44 | 19. Idx67, 6/17/2015, 8. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory | 9.489566162 | -0.489566162 | |
45 | 20. Idx68, 6/17/2015, 9. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T effector memory | 1.507625763 | 7.492374237 | |
uL 50 ng | H2O | |||
i5 short, i7 long | 21. i502_shrt-i701_long, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1 | 2.153742332 | 6.846257668 | |
22. i502_shrt-i702_long, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2 | 2.291202871 | 6.708797129 | ||
i5 short, i7 short | 23. i503_shrt-i701_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA | 0.956108278 | 8.043891722 | /4 (2.5,7.5) |
24. i503_shrt-i702_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA | 0.956108278 | 8.043891722 | /4 (2.5,7.5) | |
i5 short, i7 long | 25. i505_shrt-i701_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA | 0.956108278 | 8.043891722 | /4 (2.5,7.5) |
26. i505_shrt-i702_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA | 0.956108278 | 8.043891722 | /4 (2.5,7.5) | |
i5 long, i7 short | 27. i506_long-i701_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA | 0.956108278 | 8.043891722 | /4 (2.5,7.5) |
28. i506_long-i702_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA | 0.956108278 | 8.043891722 | /4 (2.5,7.5) | |
i5 long, i7 long | 29. i502_long-i703_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA | 0.956108278 | 8.043891722 | /4 (2.5,7.5) |
30. i502_long-i704_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA | 0.956108278 | 8.043891722 | /4 (2.5,7.5) | |
i5 short, i7 long | 31. i503_shrt-i703_long, T7-nXTv2_i5-1, 5X tn5-059, NTC | 1 | 8 | |
All RNA for samples 4, 9, 10, 12, 14, 19 was used
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.
USE nXTv2 PRIMER FOR nXTv2
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
FOR nXTv2 USE nXTv2 TRANSPOSOME FOR 3' ADDITION!!!!
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!!!!!
FOR nXTv2 METHOD USE N5XX AND N7XX PRIMERS AND INDEXES!!!
KAPA SYBR FAST qPCR mix until saturation, X35 cycles sample list 1. Idx49, 6/22/2015, 1. t7-top2, 5X tn5-059, 150 cells, mouse 1, T memory precoursor (Tmp) (CD25low CD62Lhi) 2. Idx50, 6/22/2015, 3. t7-top2, 5X tn5-059, 150 cells, mouse 1, T short lived effector (Tsle) (CD25hi CD62Llow) 3. Idx51, 6/22/2015, 5. t7-top2, 5X tn5-059, 150 cells, mouse 1, activated parental cells (CD44hi) 4. Idx52, 6/22/2015, 7. t7-top2, 5X tn5-059, 150 cells, mouse 2, T memory precoursor (Tmp) (CD25low CD62Lhi) 5. Idx53, 6/22/2015, 10. t7-top2, 5X tn5-059, 150 cells, mouse 2, T short lived effector (Tsle) (CD25hi CD62Llow) 6. Idx54, 6/22/2015, 14. t7-top2, 5X tn5-059, 150 cells, mouse 2, activated parental cells (CD44hi) 7. Idx55, 6/9/2015, 16. t7-top2, 5X tn5-059, 6 ng pure DNA 8. Idx56, 6/9/2015, 17. t7-top2, 5X tn5-059, NTC 9. Idx57, 6/9/2015, 1. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle 10. Idx58, 6/9/2015, 2. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle 11. Idx59, 6/9/2015, 3. t7-top2, 5X tn5-059, 1000 cells, day 7 Tsle 12. Idx60, 6/9/2015, 6. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp 13. Idx61,6/9/2015, 7. t7-top2, 5X tn5-059, 250 cells, day 7 Tmp 14. Idx62, 6/9/2015, 8. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp 15. Idx63, 6/17/2015, 1. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory 16. Idx64, 6/17/2015, 2. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory 17. Idx65, 6/17/2015, 3. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T central memory 18. Idx66, 6/17/2015, 7. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory 19. Idx67, 6/17/2015, 8. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory 20. Idx68, 6/17/2015, 9. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T effector memory
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 1m30s 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
- all went up. NTC is the lowest curve
File:2015-09-09 first division, day 7, day 90 T cells run.bmp
- gel after PCR addition of barcodes
File:ZhangLab 2 2015-09-09 17hr 57min-labeled.jpg File:ZhangLab 2 2015-09-09 18hr 00min-labeled.jpg
- amounts for gel size selection
- did 2 tubes of the same volumes
gel size selection amounts | intensity | ng/ul | for 80 ng | X2 |
1. Idx49, 6/22/2015, 1. t7-top2, 5X tn5-059, 150 cells, mouse 1, T memory precoursor (Tmp) (CD25low CD62Lhi) | 39239.88117 | 31.45248075 | 2.54351956 | 5.08703912 |
2. Idx50, 6/22/2015, 3. t7-top2, 5X tn5-059, 150 cells, mouse 1, T short lived effector (Tsle) (CD25hi CD62Llow) | 32547.42097 | 25.43278226 | 3.145546531 | 6.291093062 |
3. Idx51, 6/22/2015, 5. t7-top2, 5X tn5-059, 150 cells, mouse 1, activated parental cells (CD44hi) | 39874.28119 | 32.02310751 | 2.498196029 | 4.996392058 |
7. Idx55, 6/9/2015, 16. t7-top2, 5X tn5-059, 6 ng pure DNA | 42758.62127 | 34.61749838 | 0.6 | 0.6 |
8. Idx56, 6/9/2015, 17. t7-top2, 5X tn5-059, NTC | 3862.660115 | -0.368433835 | 1 | 1 |
10. Idx58, 6/9/2015, 2. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle | 34550.25103 | 27.23427718 | 2.937474693 | 5.874949386 |
14. Idx62, 6/9/2015, 8. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp | 28657.81085 | 18.08788196 | 4.97570695 | 9.9514139 |
15. Idx63, 6/17/2015, 1. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory | 26313.75078 | 16.44111556 | 5.474081104 | 10.94816221 |
19. Idx67, 6/17/2015, 8. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory | 29893.25089 | 18.95581243 | 4.747884077 | 9.495768153 |
- gel size selection
File:ZhangLab 2 2015-09-10 14hr 03min-labeled.jpg File:ZhangLab 2 2015-09-10 14hr 05min-labeled.jpg
- gel size selection validation
File:ZhangLab 2 2015-09-10 18hr 23min-labeled.jpg
future[edit]
- balh