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 | ||
Line 20: | Line 25: | ||
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 | ||
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) | 1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL) | ||
___________ | ___________ | ||
Line 56: | Line 90: | ||
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: | ||
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. | ||
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. | |||
DNA | 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 [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. | 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. | |||
* |
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.