Brandon:LabNotes/Project1/2015-9-8: Difference between revisions

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>Bsos
(Created page with "==RNA processing for rest ofjohn chang T cell samples, first cell division, day7 Tsle and Tmp, day 90 memory, == *run worked on HiSeq, will run rest of samples [http://geno...")
 
>Bsos
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  3. Idx51, 6/22/2015, 5. t7-top2, 5X tn5-059, 150 cells, mouse 1, activated parental cells (CD44hi)
  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)
  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)
  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)
  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
  7. Idx55, 6/9/2015, 16. t7-top2, 5X tn5-059, 6 ng pure DNA
Line 38: Line 38:
  19. Idx67, 6/17/2015, 8. 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
  20. Idx68, 6/17/2015, 9. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T effector memory
21. i502_shrt-i701_long, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1
22. i502_shrt-i702_long, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2
23. i503_shrt-i701_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
24. i503_shrt-i702_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
25. i505_shrt-i701_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
26. i505_shrt-i702_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
27. i506_long-i701_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
28. i506_long-i702_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
29. i502_long-i703_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
30. i502_long-i704_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
31. i503_shrt-i703_long, T7-nXTv2_i5-1, 5X tn5-059, NTC




Line 46: Line 58:
| align="center" style="background:#f0f0f0;"|'''uL 50 ng'''
| align="center" style="background:#f0f0f0;"|'''uL 50 ng'''
| align="center" style="background:#f0f0f0;"|'''H2O'''
| align="center" style="background:#f0f0f0;"|'''H2O'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| 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||
|-
| 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||
|-
| 3. Idx51, 6/22/2015, 5. t7-top2, 5X tn5-059, 150 cells, mouse 1, activated parental cells (CD44hi)||5.170801447||3.829198553||
|-
| 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||
|-
| 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||
|-
| 6. Idx54, 6/22/2015, 14. t7-top2, 5X tn5-059, 150 cells, mouse 2, activated parental cells (CD44hi)||3.027932364||5.972067636||
|-
| 7. Idx55, 6/9/2015, 16. t7-top2, 5X tn5-059, 6 ng pure DNA||1.276475454||7.723524546||/8
|-
| 8. Idx56, 6/9/2015, 17. t7-top2, 5X tn5-059, NTC||1||8||
|-
| 9. Idx57, 6/9/2015, 1. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle||10||-1||
|-
| 10. Idx58, 6/9/2015, 2. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle||10||-1||
|-
| 11. Idx59, 6/9/2015, 3. t7-top2, 5X tn5-059, 1000 cells, day 7 Tsle||1.104233808||7.895766192||
|-
| 12. Idx60, 6/9/2015, 6. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp||10||-1||
|-
| 13. Idx61,6/9/2015, 7. t7-top2, 5X tn5-059, 250 cells, day 7 Tmp||0.520745689||8.479254311||
|-
|-
| 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
| 14. Idx62, 6/9/2015, 8. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp||0.648327683||8.351672317||
|-
|-
| 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
| 15. Idx63, 6/17/2015, 1. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory||3.656925406||5.343074594||
|-
|-
| 3. Idx51, 6/22/2015, 5. t7-top2, 5X tn5-059, 150 cells, mouse 1, activated parental cells (CD44hi)||5.170801447||3.829198553
| 16. Idx64, 6/17/2015, 2. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory||4.864377142||4.135622858||
|-
|-
| 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
| 17. Idx65, 6/17/2015, 3. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T central memory||0.975440928||8.024559072||
|-
|-
| 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
| 18. Idx66, 6/17/2015, 7. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory||4.446580533||4.553419467||
|-
|-
| 6. Idx54, 6/22/2015, 14. t7-top2, 5X tn5-059, 150 cells, mouse 2, activated parental cells (CD44hi)||3.027932364||5.972067636
| 19. Idx67, 6/17/2015, 8. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory||9.489566162||-0.489566162||
|-
|-
| 7. Idx55, 6/9/2015, 16. t7-top2, 5X tn5-059, 6 ng pure DNA||1.276475454||7.723524546
| 20. Idx68, 6/17/2015, 9. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T effector memory||1.507625763||7.492374237||
|-
|-
| 8. Idx56, 6/9/2015, 17. t7-top2, 5X tn5-059, NTC||1||8
| ||||||
|-
|-
| 9. Idx57, 6/9/2015, 1. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle||10||-1
| ||uL 50 ng||H2O||
|-
|-
| 10. Idx58, 6/9/2015, 2. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle||10||-1
| 21. i502_shrt-i701_long, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1||2.153742332||6.846257668||
|-
|-
| 11. Idx59, 6/9/2015, 3. t7-top2, 5X tn5-059, 1000 cells, day 7 Tsle||1.104233808||7.895766192
| 22. i502_shrt-i702_long, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2||2.291202871||6.708797129||
|-
|-
| 12. Idx60, 6/9/2015, 6. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp||10||-1
| 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)
|-
|-
| 13. Idx61,6/9/2015, 7. t7-top2, 5X tn5-059, 250 cells, day 7 Tmp||0.520745689||8.479254311
| 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)
|-
|-
| 14. Idx62, 6/9/2015, 8. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp||0.648327683||8.351672317
| 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)
|-
|-
| 15. Idx63, 6/17/2015, 1. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory||3.656925406||5.343074594
| 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)
|-
|-
| 16. Idx64, 6/17/2015, 2. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory||4.864377142||4.135622858
| 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)
|-
|-
| 17. Idx65, 6/17/2015, 3. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T central memory||0.975440928||8.024559072
| 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)
|-
|-
| 18. Idx66, 6/17/2015, 7. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory||4.446580533||4.553419467
| 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)
|-
|-
| 19. Idx67, 6/17/2015, 8. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory||9.489566162||-0.489566162
| 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)
|-
|-
| 20. Idx68, 6/17/2015, 9. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T effector memory||1.507625763||7.492374237
| 31. i503_shrt-i703_long, T7-nXTv2_i5-1, 5X tn5-059, NTC||1||8||
|-
|-
|  
|  

Revision as of 18:23, 8 September 2015

RNA processing for rest ofjohn chang T cell samples, first cell division, day7 Tsle and Tmp, day 90 memory,

  • 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 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, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1
22. i502_shrt-i702_long, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2
23. i503_shrt-i701_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
24. i503_shrt-i702_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
25. i505_shrt-i701_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
26. i505_shrt-i702_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
27. i506_long-i701_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
28. i506_long-i702_orig, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
29. i502_long-i703_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
30. i502_long-i704_long, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
31. i503_shrt-i703_long, T7-nXTv2_i5-1, 5X tn5-059, NTC



  • Amounts of RNA and water to add for MMLV RT (50 ng RNA)
' uL 50 ng H2O '
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
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
3. Idx51, 6/22/2015, 5. t7-top2, 5X tn5-059, 150 cells, mouse 1, activated parental cells (CD44hi) 5.170801447 3.829198553
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
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
6. Idx54, 6/22/2015, 14. t7-top2, 5X tn5-059, 150 cells, mouse 2, activated parental cells (CD44hi) 3.027932364 5.972067636
7. Idx55, 6/9/2015, 16. t7-top2, 5X tn5-059, 6 ng pure DNA 1.276475454 7.723524546 /8
8. Idx56, 6/9/2015, 17. t7-top2, 5X tn5-059, NTC 1 8
9. Idx57, 6/9/2015, 1. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle 10 -1
10. Idx58, 6/9/2015, 2. t7-top2, 5X tn5-059, 100 cells, day 7 Tsle 10 -1
11. Idx59, 6/9/2015, 3. t7-top2, 5X tn5-059, 1000 cells, day 7 Tsle 1.104233808 7.895766192
12. Idx60, 6/9/2015, 6. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp 10 -1
13. Idx61,6/9/2015, 7. t7-top2, 5X tn5-059, 250 cells, day 7 Tmp 0.520745689 8.479254311
14. Idx62, 6/9/2015, 8. t7-top2, 5X tn5-059, 100 cells, day 7 Tmp 0.648327683 8.351672317
15. Idx63, 6/17/2015, 1. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory 3.656925406 5.343074594
16. Idx64, 6/17/2015, 2. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T central memory 4.864377142 4.135622858
17. Idx65, 6/17/2015, 3. t7-top2, 5X tn5-059, 1000 cells, Mouse1, day ~90 T central memory 0.975440928 8.024559072
18. Idx66, 6/17/2015, 7. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory 4.446580533 4.553419467
19. Idx67, 6/17/2015, 8. t7-top2, 5X tn5-059, 150 cells, Mouse1, day ~90 T effector memory 9.489566162 -0.489566162
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
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
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)
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)
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)
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)
31. i503_shrt-i703_long, T7-nXTv2_i5-1, 5X tn5-059, NTC 1 8


8. single strand synthesis MMLV RT (Clontech)

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

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, 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

  • PCR addition of barcodes


  • gel after PCR addition of barcodes


  • amounts for gel size selection
  • did 2 tubes of the same volumes


  • gel size selection


  • gel size selection validation


future

  • balh