Brandon:LabNotes/Project1/2013-7-10: Difference between revisions

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>Bsos
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                 DNA /5Phos/GGG]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                 DNA /5Phos/GGG]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                 cDNA  3'- CCC]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
                 cDNA  3'- CCC]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
                                                                                                     3'- GACTCGCCCGACCGTTCCGTCTGGC[GACACA]TAGAGCATACGGCAGAAGACGAAC -5' (custom barcodes) (Tm=86C)
                                                                                                     3'- GACTCGCCCGACCGTTCCGTCTGGC[GACACA]TAGAGCATACGGCAGAAGACGAAC -5' (custom NXTA barcodes) (Tm=86C)
                                                                                                     3'- GACTCGCCCGACCGTTCCGTCTGGC[------]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                     3'- GACTCGCCCGACCGTTCCGTCTGGC[------]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                                                         3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)
                                                                                                                                         3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)
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| enzyme||MMLB||Rnase H||BST 2.0||taq2x||klenow exo-||DNAPI (NEB info)
| enzyme||MMLB||Rnase H||BST 2.0||taq2x||klenow exo-||DNAPI (NEB info)
|-
|-
| reaction buffer [orig]||5X FSS||10X (B9220)||1X B pack||2X||10X RB||1X?
| reaction buffer [orig]||5X FSS||10X (B9220)||10X B pack||2X||10X RB||1X?
|-
|-
| 1X concentration||1X||1X||1X||1X||1X||1X
| 1X concentration||1X||1X||1X||1X||1X||1X
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|  
|  
|}
|}
*Polymerase incubation times/temperatures and heat inactivation information
DNAPI
30 min. at 37°C
Inactivation: 75°C for 20 min
Klenow exo-
30 minutes at 37°C un (10X reaction buffer)
Inactivation: 75°C for 20 min
BST polymerase: (10X isothermal amplification buffer)
Incubate at 65°C (for 30 mintues)
Inactivation: 80°C for 20 min
Taq2X
Incubate at 72C for 10 minutes (Tm of DNA primer is 79C) (no reaction buffer)
No heat inactivation
Rnase H  (10X reaction buffer)
20 minutes at 37°C
Inactivation: 65°C for 20 min




Line 225: Line 252:
  30 mM MgCl2
  30 mM MgCl2
  10 mM 20  mM DTT (suggest 20 mM, used 10 mM in our reactions, 2 uL of 100 mM DTT in 20 uL rxn)
  10 mM 20  mM DTT (suggest 20 mM, used 10 mM in our reactions, 2 uL of 100 mM DTT in 20 uL rxn)
   
   
  Storage buffer
  Storage buffer
Line 382: Line 408:




'''AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH AMPURE BEADS, USING RNA GENERATED FROM PURE JURKAT DNA FOR TESTING'''




 
3. single strand synthesis MMLV RT (Clontech)
12. single strand synthesis MMLV RT (Clontech)


*Followed protocol for [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/SMART_MMLV_RT SMART MMLV Reverse Transcriptase]
*Followed protocol for [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/SMART_MMLV_RT SMART MMLV Reverse Transcriptase]
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2. polymerase conditions:


  Taq2X
 
  Incubate at 72C for 8 minutes (Tm of DNA primer is 79C) (no reaction buffer)
4. 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.
 
 
 
5. Second strand synthesis: Adding sss_NPA_prmr primer and polymerases
 
a. Add 2.5 uL of sss_NPA_prmr to each reaction.
b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice.
c. Add 2 uL of dNTPs to each reaction.
d. Add polymerases etc to reactions
          DNAP1: Dilute 2X to 5U/uL and add 1 uL to the reaction. Incubate at 37C for 30 minutes
                  (stock conc. 10U/uL)
   
    Klenow exo-: Add 1 uL of 5U/uL. Incubate at 37C for 30 minutes.
   
        BST 2.0: Add 2.5 uL 10X BST buffer, dilute enzyme 2X to 4 U/ul, add 1 uL to the reaction.
                  Incubate at 65C for 30 minutes
  BST 2.0 No buf: dilute enzyme 2X to 4 U/ul, add 1 uL to the reaction. Incubate at 65C for 30 minutes
          taq2X: Add 25 uL, Incubate at 72C for 10 minutes
         
 
 
6. Clean DNA with with Zymo DNA Clean & Concentrator-5 Kit OR AMPURE beads depending. elute in 6 uL or less. (can also use qiaquick)
can quantitate with Qubit or on TBU gel.
 
 
 
 
7. Fragmenting and 3' End tagging with Custom Nextera transposome
 
1 uL nextera LMW buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
X uL Nuclase free H2O
.8 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL)
___________
5 uL total solution
 
 
 
 
 
8. 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
 
 
 
 
 
9. Fill in reaction
*Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
 
 
'''CAN TRY JUST FINISHING UP WITH TAQ2X'''
 
10. Clean DNA with with Zymo DNA Clean & Concentrator-5 Kit OR AMPURE beads depending. elute in 10 uL or less. (can also use qiaquick)
can quantitate with Qubit or on TBU gel.
 
 
11. PCR addition of barcodes '''MAKE SURE TO USE NEXTERA INDEXES!!!!!'''
Samples:
Index 65, N2 adaptor  3.  No RnIII (200 ng RNA)
Index 66, N2 adaptor  4.  5X diluted RnIII
   
   
  Klenow exo-
  KAPA SYBR FAST qPCR mix until saturation, X35 cycles
30 minutes at 37°C un (10X reaction buffer)
   
   
  BST polymerase: (10X reaction buffer)
  12.5 uL KAPA SYBR FAST qPCR mix
  Reaction Conditions
2    uL primers, 2 uL F, 2 uL R (T7-top2-PCR-iaf, OR iaf2, iaf3, and NXTA indexes PCR_R.NXTAInd[XX])
  1X Isothermal Amplification Buffer
  .5 uL H2O
  Incubate at 65°C (for 30 mintues)
  10    uL DNA template (use half RT reaction)
_____________
25 uL
   
   
  DNAP (no reaction buffer right now)
  KAPA SYBR cycles:
in 30 min. at 37°C
98C 3min, (98C for 30s, 60C for 30s, 72C for 2 min) X35, 72C for 5 min, 4C forever
   
   
Rnase H (10X reaction buffer)
*terminate before curves saturate (usually cycle 6-7)
20 minutes at 37°C
 
Heat Inactivation
 
65°C for 20 min
===Future===
 
 
 
*try experiment where adding in Rnase H, NPA primer and polymerase into the reaction at once. Next incubate at 37C for 5 minutes, and then incuvbate the reactions at suggested temperatures for the polymerases for the suggested amounts of time. Currently some protocols use this methodto reduce the number of steps.

Revision as of 02:39, 12 July 2013

Testing new method of RNA processing, No RNase III, No polyA polymerase (PAP)

  • In performing the Tn5 accessibility method, the most troubling steps are when RNase III is used for fragmentation and polyA polymerase is used to add polyA tails to the 3' end, so the T20VN_PE_R primer can be used to add adaptors/barcodes etc. Thus this protocol avoids the usage of both enzymes. RNase III does not seem to have a good shelf life and does not function correctly if it is not taken care of, and stops working approximately 3 months after purchase. PAP is a extremely robust enzyme, and adds polyA to most segments. Most of the unmapped reads are polyA tailed fragments with the sequencing reads consisting of polyA sequences.


  • Changes & testing:


    • Another robust method of generating cDNA will be used, using random hexamers for priming and MMLV for RT


    • RNA in the RNA/cDNA hybrid will be degraged with RNase H, and heated for denaturing. Next, extension with BST 2.0 or Klenow exo- will be done with a DNA primer to the 5' end.


    • DNA will will need to be fragmented, but this time a custom transpsome will be created that only has the nextera B ends on it, the 3' end of fragments were barcodes are attached.


    • BST, taq2x, klenow exo- and DNAPI will be tested for second strand synthesis, with and without Rnase H treatment.


  • In the end Fragmentation is still needed and is being performed by Nextera transposition. Other ideas for DNA fragmentation could also be entertained.



Sequences information, Using T7tspn-top2

Sequences:

5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top2)
       spcr      T7 forward    required spcr   read sequence  spcr       ME 

5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot)

 T7 description:
                                                        +1
                              CATGAGA[TTAATACGACTCACTATA|G|GGAGA]
[end sequence for optimal binding]         T7 -->          [front sequence for optimal binding]



After Annealing:

       5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’         (T7tspn-top2)
                                                         3'- TCTACACATATTCTCTGTC [Phos]-5'   (T7tspn-bot)

5'- [phos] CTGTCTCTTATACACATCT -3'
      3’-  GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTAGAGGGATATCACTCAGCATAATTAGAGTAC -5’


Fragments created after transposition:


5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3'
                                                    3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 


Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom:

NNNNN------ CTGTCTCTTATACACATCT -3'              5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’              3'- TCTACACATATTCTCTGTC ------NNNN



After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also)

5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN


NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’



After IVT, All RNA strands will have same 5' end. RNA is also purified after IVT.
First G in GGG is the +1 site for the beginning of RNA synthesis for T7.
for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator)

RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'



After single strand cDNA synthesis to form cDNA/RNA hybrid

RNA  5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'



After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer.
Extenstion with BST or klenow exo- or DNAP1 or Taq2x

DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'(Tm=79) (sss_NPA_prmr) (No Poly A)
cDNA   3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'


Now have double stranded DNA, which should be long.

DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
cDNA   3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'


normal Nextera 5' end:

5'-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3' ("A-METS" NXTA_ME_A) 
                  3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS")

after fill in:

5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 
3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'

PCR amplification:

 5'-AATGATACGGCGACCACCGA-3' (NXTA_P1)
 5'-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG-3' (NXTA_adpt1)
                             5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 
                             3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'



Custom Nextera transposome
5'- /5Phos/CTGTCTCTTATACACATCT -3' (NXTA_ME_BOT, "pMENTS")
       3'- GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'  (NXTA_ME_B, "B-METS")


                          3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS")
       5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3'  (NXTA_ME_B, "B-METS")


After custom nextera tagmentation:

DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3'
cDNA   3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'



After fill in:

DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
cDNA   3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'


Amplification:
5’- [AATGATACGGCGACCACCGA]GGG]AGATCCTCCCTCGCGCCATCAGAG     T7-top2-PCR-iaf3 (comp part (Tm=77), all (Tm=85C)
                 
    5’- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAG     T7-top2-PCR-iaf2 (comp part (Tm=73), all (Tm=83C))
                          X X 
    5’- [AATGATACGGCGACCACCGA]GATCTCTCCCTCGCGCCATCAGAGAT  T7-top2-PCR-iaf (original) (comp region 70C) (whole Tm=83C) 
                          X X XXXXX
               DNA /5Phos/GGG]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
               cDNA   3'- CCC]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[GACACA]TAGAGCATACGGCAGAAGACGAAC -5' (custom NXTA barcodes) (Tm=86C)
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[------]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                                                        3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)


buffers compatibility

company clontech enzymatics NEB NEB Epicentre Invitrogen
enzyme MMLB Rnase H BST 2.0 taq2x klenow exo- DNAPI (NEB info)
reaction buffer [orig] 5X FSS 10X (B9220) 10X B pack 2X 10X RB 1X?
1X concentration 1X 1X 1X 1X 1X 1X
pH 8.3@25C 8.3 @ 25C 8.8 @ 25C 8.6 @ 25C 7.5 7.9 @ 25C
Tris-HCl (mM) 50 50 20 10 200 10
KCl (mM) 75 75 50 50 - -
NaCl - - - - 50 50
MgCl2 (mM) 6 3 - 1.5 5 10
MgSO4 (mM) - - 2 - - -
(NH4)2SO4 (10 mM) - - 10 - - -
DTT (mM) 10 10 - - 0.5 1
Tween® 20 - - 0.10% 0.05% - -
mM dNTPs - - - 0.2 - -
Glycerol - - - 5% - -
IGEPAL® CA-630 - - - 0.08% - -
25 units/ml Taq DNA Pol - - - 25 - -




  • Polymerase incubation times/temperatures and heat inactivation information
DNAPI
30 min. at 37°C
Inactivation: 75°C for 20 min

Klenow exo-
30 minutes at 37°C un (10X reaction buffer)
Inactivation: 75°C for 20 min


BST polymerase: (10X isothermal amplification buffer)
Incubate at 65°C (for 30 mintues)
Inactivation: 80°C for 20 min

Taq2X
Incubate at 72C for 10 minutes (Tm of DNA primer is 79C) (no reaction buffer)
No heat inactivation

Rnase H  (10X reaction buffer)
20 minutes at 37°C
Inactivation: 65°C for 20 min



  • Clontech MMLV
Invitrogen
5X First-Strand Buffer
250 mM Tris-HCl (pH 8.3 at room temperature
375 mM KCl
30 mM MgCl2
10 mM	20  mM DTT (suggest 20 mM, used 10 mM in our reactions, 2 uL of 100 mM DTT in 20 uL rxn)

Storage buffer
20 mM Tris-HCl (pH 7.5)
100 mM NaCl
0.1 mM EDTA
1 mM DTT
0.01% (v/v) NP-40
50% (v/v) glycerol
  • NEB BST 2.0 Polymerase
1X Isothermal Amplification Buffer Pack:
20 mM Tris-HCl
10 mM (NH4)2SO4
50 mM KCl
2 mM MgSO4
0.1% Tween® 20
pH 8.8 @ 25°C

Storage Temperature
-20°C

Storage Conditions
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
0.1% Triton® X-100
pH 7.5 @ 25°C

Heat Inactivation
80°C for 20 min

  • NEB taq2x
Storage Temperature
-20°C
Buffer Composition
10 mM Tris-HCl
50 mM KCl
1.5 mM MgCl2
0.2 mM dNTPs
5% Glycerol
0.08% IGEPAL® CA-630
0.05% Tween® 20
25 units/ml Taq DNA Polymerase
pH 8.6@25°C

  • Epicentre klenow exo-
Storage Buffer: 
50% glycerol solution 
50 mM Tris-HCl (pH 7.5)
100 mM NaCl
1.0 mM dithiothreitol (DTT)
0.1 mM EDTA
0.1% Triton® X-100

10X reaction buffer
0.2 M Tris-HCl (pH 7.5)
50 mM MgCl2
5.0 mM DTT.


  • Invitrogen DNAPI
Unit Reaction Conditions: 
50 mM potassium phosphate (pH 7.0)
6.7 mM MgCl2
1 mM 2-mercaptoethanol
80 µg/ml templateprimer
32 µM dTTP
69 nM [3H]dTTP
and enzyme in 100 µl for 30 min. at 37°C.

NEB DNAPI
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM DTT
pH 7.9 @ 25°C

Heat Inactivation
75°C for 20 min

  • RNase H
Supplied in 
20 mM Tris-HCl
100 mM KCl
10 mM MgCl2
0.1 mM EDTA
0.1 mM DTT
50% glycerol
pH 7.9 @ 25°C 

10X RNAse H Buffer (B9220): 
500 mM Tris-HCl
750 mM KCl
30 mM MgCl2
100 mM DTT
pH 8.3 @ 25°C

  • nextera
5x Tagmentation reaction buffer 
50 mM Tris-OAc pH 8.0
25 mM Mg(OAc)2




Protocol

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

Generation of T7tspn-top2 transposome

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


Generation of custom barcode side nextera transposome

1. annealing of ME sequence to Nextera transposon sequence

    • a. Make 100 uM stock solution of (NXTA_ME_BOT, "pMENTS") and (NXTA_ME_B, "B-METS").
    • 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 Nextera transposon
1.25 uL of 100% sterile glycerol
2.50 uL of Ez-TN5 transposase
  • store at -20, is good for a year


  • check is have reagents etc


AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH AMPURE BEADS, USING RNA GENERATED FROM PURE JURKAT DNA FOR TESTING


3. 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 12.5 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.
   2  uL 5X first strand buffer
   2  uL dNTP mix
   2  uL 100 mM DTT
   1  uL N-H20
  .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



4. 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.


5. Second strand synthesis: Adding sss_NPA_prmr primer and polymerases

a. Add 2.5 uL of sss_NPA_prmr to each reaction. 

b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice.

c. Add 2 uL of dNTPs to each reaction.

d. Add polymerases etc to reactions

          DNAP1: Dilute 2X to 5U/uL and add 1 uL to the reaction. Incubate at 37C for 30 minutes
                 (stock conc. 10U/uL)

    Klenow exo-: Add 1 uL of 5U/uL. Incubate at 37C for 30 minutes.

        BST 2.0: Add 2.5 uL 10X BST buffer, dilute enzyme 2X to 4 U/ul, add 1 uL to the reaction.
                 Incubate at 65C for 30 minutes

 BST 2.0 No buf: dilute enzyme 2X to 4 U/ul, add 1 uL to the reaction. Incubate at 65C for 30 minutes

          taq2X: Add 25 uL, Incubate at 72C for 10 minutes
          


6. Clean DNA with with Zymo DNA Clean & Concentrator-5 Kit OR AMPURE beads depending. elute in 6 uL or less. (can also use qiaquick) can quantitate with Qubit or on TBU gel.



7. Fragmenting and 3' End tagging with Custom Nextera transposome

1 uL nextera LMW buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
X uL Nuclase free H2O
.8 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL) 
___________
5 uL total solution



8. 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



9. Fill in reaction

  • Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)


CAN TRY JUST FINISHING UP WITH TAQ2X

10. Clean DNA with with Zymo DNA Clean & Concentrator-5 Kit OR AMPURE beads depending. elute in 10 uL or less. (can also use qiaquick) can quantitate with Qubit or on TBU gel.


11. PCR addition of barcodes MAKE SURE TO USE NEXTERA INDEXES!!!!!

Samples:

Index 65, N2 adaptor  3.  No RnIII (200 ng RNA)
Index 66, N2 adaptor  4.  5X diluted RnIII


KAPA SYBR FAST qPCR mix until saturation, X35 cycles

12.5 uL KAPA SYBR FAST qPCR mix
2    uL primers, 2 uL F, 2 uL R (T7-top2-PCR-iaf, OR iaf2, iaf3, and NXTA indexes PCR_R.NXTAInd[XX])
.5  uL H2O
10    uL DNA template (use half RT reaction)
_____________
25 uL

KAPA SYBR cycles:
98C 3min, (98C for 30s, 60C for 30s, 72C for 2 min) X35, 72C for 5 min, 4C forever

  • terminate before curves saturate (usually cycle 6-7)


Future

  • try experiment where adding in Rnase H, NPA primer and polymerase into the reaction at once. Next incubate at 37C for 5 minutes, and then incuvbate the reactions at suggested temperatures for the polymerases for the suggested amounts of time. Currently some protocols use this methodto reduce the number of steps.