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

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===buffers compatibility===
===buffers compatibility===
*10X RNAse H Buffer (B9220)
*1X Isothermal Amplification Buffer Pack for BST 2.0 polymerase
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''company'''
| align="center" style="background:#f0f0f0;"|'''clontech'''
| align="center" style="background:#f0f0f0;"|'''enzymatics'''
| align="center" style="background:#f0f0f0;"|'''NEB'''
| align="center" style="background:#f0f0f0;"|'''NEB'''
| align="center" style="background:#f0f0f0;"|'''Epicentre'''
| align="center" style="background:#f0f0f0;"|'''Invitrogen'''
|-
| 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?
|-
| 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||-||-
|-
|
|}


*Clontech MMLV
*Clontech MMLV

Revision as of 19:46, 11 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 barcodes) (Tm=86C)
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[------]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                                                        3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)


buffers compatibility

  • 10X RNAse H Buffer (B9220)
  • 1X Isothermal Amplification Buffer Pack for BST 2.0 polymerase
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) 1X 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 - -



  • 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




12. 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


2. polymerase conditions:

Taq2X
Incubate at 72C for 8 minutes (Tm of DNA primer is 79C) (no reaction buffer)

Klenow exo-
30 minutes at 37°C un (10X reaction buffer)

BST polymerase: (10X reaction buffer)
Reaction Conditions
1X Isothermal Amplification Buffer
Incubate at 65°C (for 30 mintues)

DNAP (no reaction buffer right now)
in 30 min. at 37°C

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