Brandon:LabNotes/Project1/2015-9-8

From ZhangLabWiki
Jump to navigation Jump to search

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]


  • 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)

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