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. 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)
  ___________
  ___________
Line 56: Line 90:




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)


4. (Optional) Add 1 uL of TURBO DNase, mix well, incubate at 37C for 15 minutes.
*used 100 ng RNA for each reaction.
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.


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


  Ethanol preciptation:
9. IMPORTANT!!!
  1. Add 30 uL water to DNase-I treated transcription reaction to bring the volume to 50 uL.
*include all samples now!! (1-11)
  2. Add 3 volumes 10% EtOH, .1 volumes 3M NaOAc, and 1/300 volumes glycol Blue to the solution obtained.
second strand synthesis (qPCR) (KAPA)
    In this case:
 
    5  uL NaOAc (mix after adding)
KAPA SYBR FAST qPCR mix X35 cycles
    150 uL  100% EtOH
   
    1  uL (1/10 dilution Glycol Blue)
  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))
uL H2O
  20    uL DNA template (use whole RT reaction)
   
   
  3. Chill solution in -80 for 30 minutes, cool centrifuge to 4C
  KAPA SYBR cycles:
98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever
   
   
4. Spin at 4C for >15 minutes at max speed. Chill 75% EtOH.  
*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.
 
===Results===
 
*used GM12878 lymphocytes
*used .8 uL of transposon to save on transposome mixture. according to shendure protocol, .626 uL would be the equivalent of what they used for their assay.
 
 
*used Qubit for RNA quantitation.
*samples gave roughly the same amount of RNA regradless of starting cell amounts.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''sample#'''
| align="center" style="background:#f0f0f0;"|'''description'''
| align="center" style="background:#f0f0f0;"|'''RNA'''
| align="center" style="background:#f0f0f0;"|'''total in sample'''
|-
| 4||10000 Cells||33.4 ng/ul||400.8 ng
|-
| 5||1000 Cells||46.8 ng/ul||561.6 ng
|-
| 6||100 Cells||39.9 ng/ul||478.8 ng
|-
| 7||1000 cells control||2.75 ng/ul||33 ng
|-
| 8||pure DNA only||Out Of Range||Out Of Range
|-
| 9||1000 Cells only||3.1 ng/ul||37.2 ng
|-
| 10||N-H2O||Out Of Range||Out Of Range
|-
|
|}
 
 
 
*used random nonamer for MMLV RT for samples 4-10. used 100 ng total for RNA, except for controls where used 2.5 uL of each.
*for qPCR, used different primers for different samples
samples 4-10
T7-top2-PCR-iaf
PCR_R.N2Ind[24]
   
   
  5. Blue pellet should be visible, Discard supernatent.
  samples 1-3,11
T7-top2-PCR-iaf
N9_PE_R
   
   
  6. Wash the pellet with cold 75% EtOH
 
*curves were terminated at end of cycle 7 for samples 5,6,7 (10000,1000,100 cells respectively) and end of cycle 10 for the rest of the samples.
*samples 1-3 are pure DNA, and 4 is sample 11 (N-H2O). samples 5-11 are samples 4-10 in original list.
[[File:2012-07-02 cells testing IVT, controls, all.bmp|600px]]
 
 
*ran all samples on gel to see smear.
*only samples 4,5,6 (10000,1000,100 cells respectively) have a smear. expect to see the same smear since the same amount of input RNA for each sample was used in MMLV RT.
[[File:ZhangLab 2 2012-07-02 19hr 03min-labeled.jpg|600px]]
 
===update 7/6/2012 IVT on pure DNA samples/IVT again on cells===
 
Procedure:
*1. IVT over nite on samples 1-3,11
*2. Zymo cleaning of RNA
*3. qubit RNA quanitaton on the samples
*4. Random nonamer MMLV on samples 1-7,11
*5. PCR addition of adaptors on samples 1-7,11
 
*primers used
T7-top2-PCR-iaf
N9_PE_R
  barcodes 49-56
*barcodes used:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''sample'''
| align="center" style="background:#f0f0f0;"|'''barcode'''
|-
| 1||49
|-
| 2||50
|-
| 3||51
|-
| 4||52
|-
| 5||53
|-
| 6||54
|-
| 7||55
|-
| 11||56
|-
|
|}
 
 
 
PCR information (only half of MMLV reaction used)
10 uL sample
1  uL T7-top2-PCR-iaf
1  uL barcode
.5 uL N-H2O
12.5 uL KAPA SYBR fast
   
   
  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.
  KAPA SYBR cycles:
98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever
   
   
8. Store RNA at -80, in aliquots if desired. Aliquot in use can be at -20.
*terminate when curves saturate, (was at 8 cycles)
 
 
*qubit qunitation results, including results from 6/29
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''7/6/2012'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
| sample#||description||RNA||total in sample||||for 200 ng||water
|-
| 1||6 ng pure DNA||37.3 ng/ul||447.6 ng||||4.468275246||4.531724754
|-
| 2||600 pg pure DNA||26.6 ng/ul||319.2 ng||||6.26566416||2.73433584
|-
| 3||60 pg pure DNA||10.9 ng/ul||130.8 ng||||9||0
|-
| 11||N-free H2O||Out Of Range||Out Of Range||||#VALUE!||#VALUE!
|-
| ||||||||||||9
|-
| ||||||||||||9
|-
| ||||||||||||9
|-
| 6/29/2012||||||||||||9
|-
| sample#||description||RNA||total in sample||||5.988023952||3.011976048
|-
| 4||10000 Cells||33.4 ng/ul||400.8 ng||||||9
|-
| 5||1000 Cells||46.8 ng/ul||561.6 ng||||4.273504274||4.726495726
|-
| 6||100 Cells||39.9 ng/ul||478.8 ng||||5.012531328||3.987468672
|-
| 7||1000 cells control||2.75 ng/ul||33 ng||||72.72727273||-63.72727273
|-
| 8||pure DNA only||Out Of Range||Out Of Range||||#VALUE!||#VALUE!
|-
| 9||1000 Cells only||3.1 ng/ul||37.2 ng||||64.51612903||-55.51612903
|-
| 10||N-H2O||Out Of Range||Out Of Range||||#VALUE!||#VALUE!
|-
|
|}
 
*qubit quanitation graph
*seems like with the cells there were just more cells than I thought used in the assay. Pure DNA curve looks fine with decreasing concentration.
[[File:2012-07-05 RNA quant graph both.png]]
 
*qPCR curves, all worked as expected.
[[File:2012-07-06 qPCR transposition vs. cells after IVT-labeled.bmp|600px]]
 
 
 
*TBE gel of samples, pure DNA samples worked as expected.
[[File:ZhangLab 2 2012-07-06 16hr 53min-labeled.jpg|600px]]
 
 
 
*next
**combine samples since now barcoded, do size selection and transformation cloning to make sure have right inserts. If correct then can submit for sequencing.
 
*7/9/2012 update
 
*comibed 8.5 uL of each sample for gel size selection, since has already been barcoded. size selected for about 400 bp and about 800 bp.
[[File:ZhangLab 2 2012-07-07 16hr 22min-labeled.jpg|600px]]
 
 
 
 
*did cloning and transformation.
**used 3 uL sample for ligation, 3 uL of ligation mixture for cloning.
 
used taq2x for amplification, X29 cycles for colony PCR.
 
12.5 uL taq2X
2    uL primers, 1 uL F, 1 uL R
5.5  uL H2O
5    uL DNA template
   
   
   
taq2x cycles:
*Now can nanodrop for quanitation or run gel. fragment RNA, polyA tail addition, adapter addition to 5' and 3' ends.
95C 5min, (95C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever
 
 
*got a couple inserts.. maybe needed to stop reaction earlier for PCR? (still have fss sample left)
*sent off for sequencing, samples 1,2,4,5,10,20, 24, 27, 30, 34, 41, 43, 55, 62, 72
 
 
 
after sequencing analysis:
*sequence of clones file
[[Media:2012-07-10 custom trpn sequecing results analysis.docx]]
 
 
 
*clones stats
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''clones'''
| align="center" style="background:#f0f0f0;"|'''% of total'''
|-
| possible||13||0.168831169
|-
| have genomic insert||8||0.103896104
|-
| total clones||77||
|-
|
|}
 
 
 
*Statistics based on sample
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''transformation'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''samples'''
| align="center" style="background:#f0f0f0;"|'''validated insert'''
|-
| 3||~800 bp gel cut-1||1-11||2
|-
| 4||~800 bp gel cut-2||12-22||1
|-
| 3||~800 bp gel cut-1||23-32||
|-
| 4||~800 bp gel cut-2||34-44||2
|-
| 1-1||~400 bp gel cut-1||45-55||1
|-
| 2-1||~400 bp gel cut-2||56-66||1
|-
| 2-1LT2||~400 bp gel cut-2-1||67-72||1
|-
| 1-1LT2||~400 bp gel cut-1-1||73-77||
|-
| ||||total||8
|-
|
|}
 
[[File:ZhangLab 2 2012-07-09 13hr 21min-labeled.jpg|600px]]
[[File:ZhangLab 2 2012-07-09 13hr 22min-labeled.jpg|600px]]
[[File:ZhangLab 2 2012-07-10 12hr 04min-labeled.jpg|600px]]
[[File:ZhangLab 2 2012-07-10 12hr 06min-labeled.jpg|600px]]
[[File:ZhangLab 2 2012-07-10 14hr 30min-labeled.jpg|600px]]
[[File:ZhangLab 2 2012-07-10 14hr 31min-labeled.jpg|600px]]
[[File:ZhangLab 2 2012-07-10 14hr 38min-labeled.jpg|600px]]
 
 
*ran TBU gel to make sure long double stranded products were being obtained still after PCR amplification and not balls or short DNAs.
[[File:ZhangLab 2 2012-07-09 16hr 37min-labeled.jpg|600px]]
 
===conclusions===
 
*During Qubit analysis, rougly the same amount of RNA was obtained from each sample after overnight IVT. unsure why, and the 10,000 cell sample gave the least amount but only by 100 nanograms. 
**It could be the amount of transposition occuring was the limiting factor.. use more transposon to fragment DNA? 
**Or IVT reaction could have saturated or used up limiting nucleotide/reagent by the time it has synthesized that much RNA.
*pure DNA, and nuclease free water were out of range, and 1000 cell controls had a minimal amount of RNA which figures since cells have RNA in them.
 
 
*Random nonamer binding did not work for samples where only transposition took place, either that or not enough DNA. can't see smears when used 5 ng/ul or DNA per sample when testing for tranposition with TBU gel.
 
 
 
===future===
 
*sequencing result from above data and experiment are on [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-7-20 sequencing data anlysis, tagmentation/IVT]
 
 
 
*half of the pure DNA samples are left, will use the rest for IVT amplification, then clean with Zymo RNA cleaning kit, quant with qubit, then MMLV RT, and PCR with adaptors with different index numbers.
 
 
*use rest of IVT RNA from tagmentation on cells and do MMLV RT, PCR with adaptors with different index numbers.
 
 
*use ~200 ng or the rest of the IVT RNA for MMLV RT to maximize input coverage.
 
 
*after barcode samples, then can do size selection and cloning/transformation to show that correct inserts are being obtained. can also combine all the samples since they will all have different barcodes.
 
 
*can then submit for sequencing. Can see if pure DNA library is randomly distributed while libraries from cells center over accessible regions. (can compare to UCSC genome browser GM12878 line again).

Latest revision as of 23:34, 23 August 2012

Measuring tagmentation efficiency cells versus pure DNA[edit]

  • 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[edit]

  • 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


  • used 100 ng RNA for each reaction.
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.

Results[edit]

  • used GM12878 lymphocytes
  • used .8 uL of transposon to save on transposome mixture. according to shendure protocol, .626 uL would be the equivalent of what they used for their assay.


  • used Qubit for RNA quantitation.
  • samples gave roughly the same amount of RNA regradless of starting cell amounts.
sample# description RNA total in sample
4 10000 Cells 33.4 ng/ul 400.8 ng
5 1000 Cells 46.8 ng/ul 561.6 ng
6 100 Cells 39.9 ng/ul 478.8 ng
7 1000 cells control 2.75 ng/ul 33 ng
8 pure DNA only Out Of Range Out Of Range
9 1000 Cells only 3.1 ng/ul 37.2 ng
10 N-H2O Out Of Range Out Of Range


  • used random nonamer for MMLV RT for samples 4-10. used 100 ng total for RNA, except for controls where used 2.5 uL of each.
  • for qPCR, used different primers for different samples
samples 4-10
T7-top2-PCR-iaf
PCR_R.N2Ind[24]

samples 1-3,11
T7-top2-PCR-iaf
N9_PE_R

  • curves were terminated at end of cycle 7 for samples 5,6,7 (10000,1000,100 cells respectively) and end of cycle 10 for the rest of the samples.
  • samples 1-3 are pure DNA, and 4 is sample 11 (N-H2O). samples 5-11 are samples 4-10 in original list.

File:2012-07-02 cells testing IVT, controls, all.bmp


  • ran all samples on gel to see smear.
  • only samples 4,5,6 (10000,1000,100 cells respectively) have a smear. expect to see the same smear since the same amount of input RNA for each sample was used in MMLV RT.

File:ZhangLab 2 2012-07-02 19hr 03min-labeled.jpg

update 7/6/2012 IVT on pure DNA samples/IVT again on cells[edit]

Procedure:

  • 1. IVT over nite on samples 1-3,11
  • 2. Zymo cleaning of RNA
  • 3. qubit RNA quanitaton on the samples
  • 4. Random nonamer MMLV on samples 1-7,11
  • 5. PCR addition of adaptors on samples 1-7,11
  • primers used
T7-top2-PCR-iaf
N9_PE_R
barcodes 49-56
  • barcodes used:
sample barcode
1 49
2 50
3 51
4 52
5 53
6 54
7 55
11 56


PCR information (only half of MMLV reaction used)

10 uL sample 
1  uL T7-top2-PCR-iaf
1  uL barcode
.5 uL N-H2O
12.5 uL KAPA SYBR fast

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, (was at 8 cycles)


  • qubit qunitation results, including results from 6/29
7/6/2012 ' ' ' ' ' '
sample# description RNA total in sample for 200 ng water
1 6 ng pure DNA 37.3 ng/ul 447.6 ng 4.468275246 4.531724754
2 600 pg pure DNA 26.6 ng/ul 319.2 ng 6.26566416 2.73433584
3 60 pg pure DNA 10.9 ng/ul 130.8 ng 9 0
11 N-free H2O Out Of Range Out Of Range #VALUE! #VALUE!
9
9
9
6/29/2012 9
sample# description RNA total in sample 5.988023952 3.011976048
4 10000 Cells 33.4 ng/ul 400.8 ng 9
5 1000 Cells 46.8 ng/ul 561.6 ng 4.273504274 4.726495726
6 100 Cells 39.9 ng/ul 478.8 ng 5.012531328 3.987468672
7 1000 cells control 2.75 ng/ul 33 ng 72.72727273 -63.72727273
8 pure DNA only Out Of Range Out Of Range #VALUE! #VALUE!
9 1000 Cells only 3.1 ng/ul 37.2 ng 64.51612903 -55.51612903
10 N-H2O Out Of Range Out Of Range #VALUE! #VALUE!
  • qubit quanitation graph
  • seems like with the cells there were just more cells than I thought used in the assay. Pure DNA curve looks fine with decreasing concentration.

File:2012-07-05 RNA quant graph both.png

  • qPCR curves, all worked as expected.

File:2012-07-06 qPCR transposition vs. cells after IVT-labeled.bmp


  • TBE gel of samples, pure DNA samples worked as expected.

File:ZhangLab 2 2012-07-06 16hr 53min-labeled.jpg


  • next
    • combine samples since now barcoded, do size selection and transformation cloning to make sure have right inserts. If correct then can submit for sequencing.
  • 7/9/2012 update
  • comibed 8.5 uL of each sample for gel size selection, since has already been barcoded. size selected for about 400 bp and about 800 bp.

File:ZhangLab 2 2012-07-07 16hr 22min-labeled.jpg



  • did cloning and transformation.
    • used 3 uL sample for ligation, 3 uL of ligation mixture for cloning.

used taq2x for amplification, X29 cycles for colony PCR.

12.5 uL taq2X
2    uL primers, 1 uL F, 1 uL R
5.5  uL H2O
5    uL DNA template

taq2x cycles:
95C 5min, (95C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever


  • got a couple inserts.. maybe needed to stop reaction earlier for PCR? (still have fss sample left)
  • sent off for sequencing, samples 1,2,4,5,10,20, 24, 27, 30, 34, 41, 43, 55, 62, 72


after sequencing analysis:

  • sequence of clones file

Media:2012-07-10 custom trpn sequecing results analysis.docx


  • clones stats
' clones % of total
possible 13 0.168831169
have genomic insert 8 0.103896104
total clones 77


  • Statistics based on sample
transformation ' samples validated insert
3 ~800 bp gel cut-1 1-11 2
4 ~800 bp gel cut-2 12-22 1
3 ~800 bp gel cut-1 23-32
4 ~800 bp gel cut-2 34-44 2
1-1 ~400 bp gel cut-1 45-55 1
2-1 ~400 bp gel cut-2 56-66 1
2-1LT2 ~400 bp gel cut-2-1 67-72 1
1-1LT2 ~400 bp gel cut-1-1 73-77
total 8

File:ZhangLab 2 2012-07-09 13hr 21min-labeled.jpg File:ZhangLab 2 2012-07-09 13hr 22min-labeled.jpg File:ZhangLab 2 2012-07-10 12hr 04min-labeled.jpg File:ZhangLab 2 2012-07-10 12hr 06min-labeled.jpg File:ZhangLab 2 2012-07-10 14hr 30min-labeled.jpg File:ZhangLab 2 2012-07-10 14hr 31min-labeled.jpg File:ZhangLab 2 2012-07-10 14hr 38min-labeled.jpg


  • ran TBU gel to make sure long double stranded products were being obtained still after PCR amplification and not balls or short DNAs.

File:ZhangLab 2 2012-07-09 16hr 37min-labeled.jpg

conclusions[edit]

  • During Qubit analysis, rougly the same amount of RNA was obtained from each sample after overnight IVT. unsure why, and the 10,000 cell sample gave the least amount but only by 100 nanograms.
    • It could be the amount of transposition occuring was the limiting factor.. use more transposon to fragment DNA?
    • Or IVT reaction could have saturated or used up limiting nucleotide/reagent by the time it has synthesized that much RNA.
  • pure DNA, and nuclease free water were out of range, and 1000 cell controls had a minimal amount of RNA which figures since cells have RNA in them.


  • Random nonamer binding did not work for samples where only transposition took place, either that or not enough DNA. can't see smears when used 5 ng/ul or DNA per sample when testing for tranposition with TBU gel.


future[edit]


  • half of the pure DNA samples are left, will use the rest for IVT amplification, then clean with Zymo RNA cleaning kit, quant with qubit, then MMLV RT, and PCR with adaptors with different index numbers.


  • use rest of IVT RNA from tagmentation on cells and do MMLV RT, PCR with adaptors with different index numbers.


  • use ~200 ng or the rest of the IVT RNA for MMLV RT to maximize input coverage.


  • after barcode samples, then can do size selection and cloning/transformation to show that correct inserts are being obtained. can also combine all the samples since they will all have different barcodes.


  • can then submit for sequencing. Can see if pure DNA library is randomly distributed while libraries from cells center over accessible regions. (can compare to UCSC genome browser GM12878 line again).