Brandon:LabNotes/Project1/2012-6-29: Difference between revisions

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>Bsos
(Created page with "==Measuring tagmentation efficiency cells versus pure DNA== *procotols from shendure paper, [http://genome.cshlp.org/content/early/2012/03/30/gr.136242.111?top=1 shendure pa...")
 
>Bsos
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*using T7-top2 for transposition reactions [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-5-7#Summary_for_using_with_T7tspn-top2: t7tspn-top2]
*using T7-top2 for transposition reactions [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-5-7#Summary_for_using_with_T7tspn-top2: t7tspn-top2]
*goal is to compare efficiency of DNA accessibility across different cellular concentrations. Also to test efficiency of tagmentation with custom transposon/transposome on pure DNA, and efficiency of amplification from IVT.
*goal is to compare efficiency of DNA accessibility across different cellular concentrations. Also to test efficiency of tagmentation with custom transposon/transposome on pure DNA, and efficiency of amplification from IVT.
**Using GM12878 cells.


If need to make more transposome.
 
 
===Protocol===
*If need to make more transposome, do first 2 steps.
1. annealing of ME sequence to T7 transposon sequence
1. annealing of ME sequence to T7 transposon sequence
**a. Make 100 uM stock solution of T7tspn-top2 and T7tspn-bot.
**a. Make 100 uM stock solution of T7tspn-top2 and T7tspn-bot.
**b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
**b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
**c. cool to RT at 0.1 C/s
**c. cool to RT at 0.1 C/s




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3. Lyse cells with lysis buffer
  4. 10,000 cells, GM12878
  5. 1,000  cells, GM12878
  6. 100    cells, GM12878


4. transposition reaction, using (T7tspn-top2)
3. Prepare samples, lyse cells with lysis buffer
 
  Samples:
  Samples:
  1. 6 ng 218HFF purified DNA
  1. 6 ng 218HFF purified DNA
Line 33: Line 35:
  5. 1,000  cells
  5. 1,000  cells
  6. 100    cells
  6. 100    cells
  7. without transposome complex
  7. 1000 cells without transposome complex
  8. pure DNA only
  8. pure DNA only (6 ng)
  9. 1000 cells lysed DNA only
  9. 1000 cells lysed DNA only
  10. Nuclease free H20 only
  10. Nuclease free H20 only (cells)
11. Nuclease free H20 only  (pure DNA)
 
 
*Make 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.
*Prepare aliquots of lymphocytes GM12878 (have GM20431, GM12878 and MEFs) that contain 10000, 1000, 100 cells.
**spin down cells to concentrate them as necessary.
*Prepare 2X LB from 10X buffer. mineral oil optional.
*if needed make 4 or 6 uL solutions. keep 1:1 ratio of cells:lysis buffer
 
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''4. 10000'''
| align="center" style="background:#f0f0f0;"|'''5. 1000'''
| align="center" style="background:#f0f0f0;"|'''6. 100'''
| align="center" style="background:#f0f0f0;"|'''7. 1000C'''
| align="center" style="background:#f0f0f0;"|'''9. 1000C'''
| align="center" style="background:#f0f0f0;"|'''10. NTC'''
| align="center" style="background:#f0f0f0;"|'''11. NTC'''
|-
| cells||1ul||1ul||1ul||1ul||1ul||1ul PBS||1ul PBS||
|-
| 2X LB||1ul||1ul||1ul||1ul||1ul||1ul||1ul||
|-
|
|}
 
*incubate at 37C for 30 mins.
 
 
 
4. transposition reaction, using (T7tspn-top2)


*add the below into one tube and incubate for 8 minutes at 55C.
*add the below into one tube and incubate for 8 minutes at 55C.
  1 uL nextera HMW buffer
  1 uL nextera HMW buffer
  1 uL genomic DNA (5 ng Jurkat DNA)
  2 uL lysed/pure genomic DNA (X ng/pg DNA)
  2 uL Nuclease free water
  1 uL Nuclease free water
  1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL)
  1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL)
  ___________
  ___________
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4. Protease digestion of transposase, protease inactivation
5. Protease digestion of transposase, protease inactivation
  To each tube, add:
  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 [])
  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 [])
Line 62: Line 96:




5. Fill in reaction
6. Fill in reaction
*Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
*Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
*IVT on filled in reaction and not filled in reaction in to show fill-in is working.
*IVT on filled in reaction and not filled in reaction in to show fill-in is working.




6. should now be ready for IVT.
7. IMPORTANT!!
*samples 1-3, 11 not included in IVT
*use samples 4-10 for IVT
Maxiscript (Ambion) T7 Protocol, IVT


===Maxiscript (Ambion) T7 Protocol===
*DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.


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!!!!
  bring to 20 uL with Nuclease free water
  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
b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)
c. Clean RNA with with Zymo RNA Clean & Concentrator-5 Kit
   
*Now can use nanodrop or qu-bit for quantitation or run gel.


1. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) on ice, but keep 10X transcription buffer at room temp


2. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
bring to 20 uL with Nuclease free water
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


8. binding of random nonamer and MMLV RT (Clontech)
*Followed protocol for [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/SMART_MMLV_RT SMART MMLV Reverse Transcriptase] (except with random nonamers) WITH MODIFICATIONS!!!
**higher denaturing temperature since RNA-RNA hybrids
**incubation times for random nonamers


3. Incubate reactions at 37C overnight. (>10 uM limiting nucleotide)
for 20 uL reaction
a. use N9_PE_R, mix 2.5 uL of 20 uM N9_PE_R with RNA sample. Bring to final volume of 11.5
    with Nuclease free H2O
    Mix well!!
b. Heat at 85C for 3 minutes. Immediately cool on ice.
c. Add the following to the reaction. (mix before adding MMLV)
    4  uL 5X first strand buffer
    2  uL dNTP mix
    2  uL 100 uM DTT
  .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  _______
  20 uL total
d. Incuvate at 16C for 10 minutes, then 42C for 60 minutes.
e. Terminate the reaction by heating at 70C for 10 minutes.


4. (Optional) Add 1 uL of TURBO DNase, mix well, incubate at 37C for 15 minutes.


5. (Optional) Add 1uL of 0.5 M EDTA to stop the reaction


6. Remove unincorporated nucleotides for quantitation. (ethanol precipitation or column precipitation). Can also run TBU gel to view sample and controls
9. IMPORTANT!!!
*include all samples now!! (1-11)
second strand synthesis (qPCR) (KAPA)


  Ethanol preciptation:
  KAPA SYBR FAST qPCR mix X35 cycles
1. Add 30 uL water to DNase-I treated transcription reaction to bring the volume to 50 uL.
2. Add 3 volumes 10% EtOH, .1 volumes 3M NaOAc, and 1/300 volumes glycol Blue to the solution obtained.
    In this case:
    5  uL NaOAc (mix after adding)
    150 uL  100% EtOH
    1  uL (1/10 dilution Glycol Blue)
   
   
  3. Chill solution in -80 for 30 minutes, cool centrifuge to 4C
  25 uL KAPA SYBR
4    uL primers, 1 uL F, 1 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX] (23,24))
1  uL H2O
20    uL DNA template (use whole RT reaction)
   
   
  4. Spin at 4C for >15 minutes at max speed. Chill 75% EtOH.
  KAPA SYBR cycles:
98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever
   
   
5. Blue pellet should be visible, Discard supernatent.
*terminate when curves saturate
 
6. Wash the pellet with cold 75% EtOH
 
10.qiaquick cleanup or beads purification
7. To completely remove incorporated NTPs, resuspend the pellet in 50 uL nuclease free water and repeat steps 1-6. Otherwise resuspend with nuclease free water.
*run on gel or w/e
*run qiaquick to clean sample before performing qPCR.
8. Store RNA at -80, in aliquots if desired. Aliquot in use can be at -20.
**6 enzymes from 6 different reactions in there already.
   
*Now can nanodrop for quanitation or run gel. fragment RNA, polyA tail addition, adapter addition to 5' and 3' ends.

Revision as of 01:11, 29 June 2012

Measuring tagmentation efficiency cells versus pure DNA

  • procotols from shendure paper, shendure paper transposition
  • using T7-top2 for transposition reactions t7tspn-top2
  • goal is to compare efficiency of DNA accessibility across different cellular concentrations. Also to test efficiency of tagmentation with custom transposon/transposome on pure DNA, and efficiency of amplification from IVT.
    • Using GM12878 cells.


Protocol

  • If need to make more transposome, do first 2 steps.

1. annealing of ME sequence to T7 transposon sequence

    • a. Make 100 uM stock solution of T7tspn-top2 and T7tspn-bot.
    • b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
    • c. cool to RT at 0.1 C/s


2. transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 20 minutes at RT
1.25 uL of annealed transposon
1.25 uL of 100% sterile glycerol
2.50 uL of Ez-TN5 transposase
  • store at -20, is good for a year


3. Prepare samples, lyse cells with lysis buffer

Samples:
1. 6 ng 218HFF purified DNA
2. 600 pg 218HFF purified DNA
3. 60  pg 218HFF purified DNA
4. 10,000 cells 
5. 1,000  cells
6. 100    cells
7. 1000 cells without transposome complex
8. pure DNA only (6 ng)
9. 1000 cells lysed DNA only
10. Nuclease free H20 only (cells)
11. Nuclease free H20 only  (pure DNA)


  • Make 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.
  • Prepare aliquots of lymphocytes GM12878 (have GM20431, GM12878 and MEFs) that contain 10000, 1000, 100 cells.
    • spin down cells to concentrate them as necessary.
  • Prepare 2X LB from 10X buffer. mineral oil optional.
  • if needed make 4 or 6 uL solutions. keep 1:1 ratio of cells:lysis buffer


' 4. 10000 5. 1000 6. 100 7. 1000C 9. 1000C 10. NTC 11. NTC
cells 1ul 1ul 1ul 1ul 1ul 1ul PBS 1ul PBS
2X LB 1ul 1ul 1ul 1ul 1ul 1ul 1ul
  • incubate at 37C for 30 mins.


4. transposition reaction, using (T7tspn-top2)

  • add the below into one tube and incubate for 8 minutes at 55C.
1 uL nextera HMW buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
1 uL Nuclease free water
1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL)
___________
5 uL total solution
method used in shendure paper:
4 uL nextera HMW buffer
X uL genomic DNA at prepared quantities
X uL Nuclease free water
______
17.5 uL total solution

add 2.5 uL of prepared transposomes


5. 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


6. Fill in reaction

  • Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
  • IVT on filled in reaction and not filled in reaction in to show fill-in is working.


7. IMPORTANT!!

  • samples 1-3, 11 not included in IVT
  • use samples 4-10 for IVT

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!!!!
  bring to 20 uL with Nuclease free water
  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


b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)


c. Clean RNA with with Zymo RNA Clean & Concentrator-5 Kit

   
  • Now can use nanodrop or qu-bit for quantitation or run gel.


8. binding of random nonamer and MMLV RT (Clontech)

  • Followed protocol for SMART MMLV Reverse Transcriptase (except with random nonamers) WITH MODIFICATIONS!!!
    • higher denaturing temperature since RNA-RNA hybrids
    • incubation times for random nonamers
for 20 uL reaction

a. use N9_PE_R, mix 2.5 uL of 20 uM N9_PE_R with RNA sample. Bring to final volume of 11.5
   with Nuclease free H2O
   Mix well!!

b. Heat at 85C for 3 minutes. Immediately cool on ice.

c. Add the following to the reaction. (mix before adding MMLV)
   4  uL 5X first strand buffer
   2  uL dNTP mix
   2  uL 100 uM DTT
  .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  _______
  20 uL total

d. Incuvate at 16C for 10 minutes, then 42C for 60 minutes.

e. Terminate the reaction by heating at 70C for 10 minutes.


9. IMPORTANT!!!

  • include all samples now!! (1-11)

second strand synthesis (qPCR) (KAPA)

KAPA SYBR FAST qPCR mix X35 cycles

25 uL KAPA SYBR
4    uL primers, 1 uL F, 1 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX] (23,24))
1  uL H2O
20    uL DNA template (use whole RT reaction)

KAPA SYBR cycles:
98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever

  • terminate when curves saturate


10.qiaquick cleanup or beads purification

  • run on gel or w/e
  • run qiaquick to clean sample before performing qPCR.
    • 6 enzymes from 6 different reactions in there already.