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

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>Bsos
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  Amplification:
  Amplification (barcode and ILA adaptor addition):
  5’- [AATGATACGGCGACCACCGA]GGG]AGATCCTCCCTCGCGCCATCAGAG    T7-top2-PCR-iaf3 (comp part (Tm=77), all (Tm=85C)
  5’- [AATGATACGGCGACCACCGA]GGG]AGATCCTCCCTCGCGCCATCAGAG    T7-top2-PCR-iaf3 (comp part (Tm=77), all (Tm=85C)
                    
                    
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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 NXTA barcodes) (Tm=86C)
                                                                                                     3'- GACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' (custom NXTA barcodes) (Tm=86C)
                                                                                                     3'- GACTCGCCCGACCGTTCCGTCTGGC[------]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                     3'- GACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                                                         3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)
                                                                                                                                         3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)
Read Primers:
 
Using T7-top2-PCR-iaf2 for amp.
                                          (Nextera_R1)
                          5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'
                                        (T7tspn-Read1)                                  (Nextera_Ind index read primer)
                          5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'      5'- CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG -3'
5’- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG[XXXXXX]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- [TTACTATGCCGCTGGTGGCT]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5'





Revision as of 23:15, 11 February 2014

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- etc will be done with a DNA primer to the 5' end.


    • DNA 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, and BST without adding BST buffer tot he reaction.


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



Graphic overview

File:Picture1-noRnIIInoPAP graphic overview.png


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 (barcode and ILA adaptor addition):
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[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' (custom NXTA barcodes) (Tm=86C)
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                                                        3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)

Read Primers:
 
Using T7-top2-PCR-iaf2 for amp.

                                         (Nextera_R1)
                         5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'
                                        (T7tspn-Read1)                                   (Nextera_Ind index read primer)
                         5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'      5'- CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG -3'
5’- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG[XXXXXX]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- [TTACTATGCCGCTGGTGGCT]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGTCTGGC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5'






buffers compatibility

company clontech enzymatics NEB NEB Epicentre Invitrogen
enzyme MMLV 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 information, incubation times/temperatures and heat inactivation information
DNAPI
some strand displacement
5´-3´ and 3´-5´ exodeoxyribonuclease activities.
30 min. at 37°C
Inactivation: 75°C for 20 min

Klenow exo-
Moderate strand displacement activity 
No 5´→ 3´ exonuclease activity and has mutations (D355A, E357A) which abolish the 3´→ 5´ exonuclease activity
30 minutes at 37°C un (10X reaction buffer)
Inactivation: 75°C for 20 min


BST polymerase: (10X isothermal amplification buffer)
strong strand displcement activity
No 5´→3´ exonuclease activity. 
Incubate at 65°C (for 30 mintues)
Inactivation: 80°C for 20 min

Taq2X
some strand displacement activity
5´ flap endonuclease activity (4,5) (degrades ssDNA displaced strand)
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



Before Starting Protocol

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • T7-top2 transposomes
  • custom nextera transposomes
  • transposase/transposome
  • IVT reaction mixture
  • cells etc
  • Rnase H, random hexamers, protease, AMPURE beads ,ATP, taq polymerase, other polymerases (BST, klewnow exo-, DNAPI), other primers


  • 2. samples this time
Index 49, NXTA adaptor, 1. DNAPI
Index 50, NXTA adaptor, 2. Klenow exo-
Index 51, NXTA adaptor, 3. BST 2.0
Index 52, NXTA adaptor, 4. BST 2.0 no buf
Index 53, NXTA adaptor, 5. taq2x
Index 54, NXTA adaptor, 6. NTC



Protocol

  • If need to make more transposome, do first 2 steps. If not goto step 3.

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 (3' side of fragments) 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



3. Prepare samples, lyse cells with lysis buffer

Samples:


  • 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.
  • spin down cells to concentrate them as necessary. Aim for X number of cells (or pure genomic DNA) in 1 uL so that 1 uL of 2X lysis buffer can be used in the reaction.
  • Prepare 2X LB from 10X buffer. mineral oil optional.
  • if needed make 4 or 6 uL solutions. ensure that lysis buffer is 1X in as the final concentration.


  • incubate the mixtures at 37C for 30 mins.


4. transposition reaction, using (T7tspn-top2)


  • add the below into one tube and incubate for 10 minutes at 55C.
1 uL nextera LMW buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
1.2 uL Nuclease free water
.8 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)


7. 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)



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


Samples this time:

Index 49, NXTA adaptor, 1. DNAPI
Index 50, NXTA adaptor, 2. Klenow exo-
Index 51, NXTA adaptor, 3. BST 2.0
Index 52, NXTA adaptor, 4. BST 2.0 no buf
Index 53, NXTA adaptor, 5. taq2x
Index 54, NXTA adaptor, 6. NTC


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 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



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.

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
          
            NTC: Just add 1 uL of Klenow exo-


11. Clean DNA with with Zymo DNA Clean & Concentrator-5 Kit OR AMPURE beads depending. elute in 6 uL or less. (can also use qiaquick) USED AMPURE DNA BEADS AT 1:1.8X RATIO 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.



12. 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


  • Incubate at 55C for 10 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)


CAN TRY JUST FINISHING UP WITH TAQ2X (DID THAT THIS TIME, ADDED 12.5 UL OF 1X TAQ2X TO SOLUTION AND STOPPED AT END OF 6TH CYCLE. ADDED SYBR GREEN .075X FINAL CONCENTRATION IN SOLUTION

15. 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.


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

Samples this time:
Index 49, NXTA adaptor, 1. DNAPI
Index 50, NXTA adaptor, 2. Klenow exo-
Index 51, NXTA adaptor, 3. BST 2.0
Index 52, NXTA adaptor, 4. BST 2.0 no buf
Index 53, NXTA adaptor, 5. taq2x
Index 54, NXTA adaptor, 6. NTC

USED T7-top2-PCR-iaf (7/10/13) (IAF2 MIGHT GIVE BETTER AMPLIFICATION

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)



Results

  • Had leftover RNA generated from transposition on pure Jurkat DNA. Used sample #2 after IVT but before purification, has been frozen at -20C. Purified with AMPURE RNA beads, adding 30 uL to solution and 1:1.8X ratio of sample:beads.
  • flow though has most of the transposon fragment which is good.
  • quantification of RNA from gel
' ng/ul intensity ' '
5 2504.210075
2.5 1036.780031 in 10 uL for 200 ng
2013-5-10 pure Jurkat DNA elution 158.6959685 92719.44276 1586.959685 1.260271461
2013-5-10 pure Jurkat DNA on beads 112.9738386 65881.83196 1129.738386

File:ZhangLab 2 2013-07-10 14hr 59min-labeled.jpg





  • TBE gel after second strand synthesis and AMPURE DNA beads purification
  • look as expected, similar to TBU gel on IVT RNA.
  • eluted samples in 20 uL N-H2O, concentrated to 10 uL, ran gel on 1 uL of the sample then.
  • qunaitation of samples:
' ng/ul intensity '
5 5401.670161
2.5 3596.240107 in 10 uL
Index 49, NXTA adaptor, 1. DNAPI 60.64528949 45587.14136 606.4528949
Index 50, NXTA adaptor, 2. Klenow exo- 77.69815224 57902.24173 776.9815224
Index 51, NXTA adaptor, 3. BST 2.0 75.27544685 56152.63167 752.7544685
Index 52, NXTA adaptor, 4. BST 2.0 no buf 77.8642761 58022.21173 778.642761
Index 53, NXTA adaptor, 5. taq2x 50.21377179 38053.79113 502.1377179
Index 54, NXTA adaptor, 6. NTC 0

File:ZhangLab 2 2013-07-12 18hr 54min-labeled.jpg



  • qPCR from barcode addition. curves didn't go up, but added in SYBR green manually and might have done that incorrectly. Also didn't clean reaction since the transposition reaction so that may have contributed to SYBR green not working correctly. Gel after barcode addition looks good.
  • samples in order
Index 49, NXTA adaptor, 1. DNAPI
Index 50, NXTA adaptor, 2. Klenow exo-
Index 51, NXTA adaptor, 3. BST 2.0
Index 52, NXTA adaptor, 4. BST 2.0 no buf
Index 53, NXTA adaptor, 5. taq2x
Index 54, NXTA adaptor, 6. NTC

File:2013-07-12 No Rnase III no PAP protocol testing.bmp




  • after custom nextera tagmentation and barcode addition
  • when compared to gel after second strand synthesis, can definetly see high molecular weight fragments have been reduced, and there are more fragments between ~200-400 bp.

File:ZhangLab 2 2013-07-15 11hr 13min-labeled.jpg



  • amounts used for gel size selection
' ng/ul intensity ' '
7/15/2013 5 6162.250184
2.5 2898.130086 for 100 ng
Index 49, NXTA adaptor, 1. DNAPI 9.098669779 11513.67034 10.99061758
Index 50, NXTA adaptor, 2. Klenow exo- 13.92165423 17810.79053 7.183054424
Index 51, NXTA adaptor, 3. BST 2.0 13.49057326 17247.95051 7.412583442
Index 52, NXTA adaptor, 4. BST 2.0 no buf 13.92723766 17818.08053 7.180174735
Index 53, NXTA adaptor, 5. taq2x 10.32603734 13116.18039 9.68425706
Index 54, NXTA adaptor, 6. NTC
100
ng total 500
sum 42.45068724
dye to add 8.254300296
per well 12.67624688


  • gel size cut validation

File:ZhangLab 2 2013-07-15 12hr 59min.jpg



  • gel size selection validation of size range

File:ZhangLab 2 2013-07-15 16hr 33min.jpg


Future

  • try experiment where adding in Rnase H, NPA primer and polymerase into the reaction at once. Then 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.


  • UPDATE AUG 7, 2013
  • Stumbled upon a paper in genome research Transposase mediated construction of RNA-seq libraries that describes generating sequencing libraries though using nextera transposons, for polyA selected RNA. some considerations can be taken from the paper.
  • 1. Random hexamer single strand DNA synthesis is basically the same.
  • 2. Second strand synthesis: can use this protocol instead, but seems negligible since for my method a RNA is wanted to be highly degraded, primer is added, and extension is wanted from that primer (sss_NPA_prmr)
20 uL sample (all sample)
16 uL water
5 uL 10X synthesis strand buffer ((500 mM Tris-HCl at pH 7.8, 50 mM MgCl2, 10 mM DTT))
3 uL 10 mM dNTPs
1 uL Rnase H
5 uL DNA polymerase I (10 u/Ul)
Reactions were incubated at 16C for 2.5 hours. (Rnase H nicks, and DNAP extends DNA from the nicks)
  • 3. Mention in methods that using 75 ng or less of double-stranded cDNA in the transposition reaction is important and larger amounts of cDNA results in large >1000 bp insert sizes. Can take this into consideration when running assay again. Can titrate amounts of RNA used for transposition and see what gives better sequencing results.