Brandon:LabNotes/Project1/2012-8-6

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Custom transposon with IVT amplification, round 2

  • comparing custom transposon to nextera tagmentation more effectively since using the same cell line, GM12878. Before nextera tagmentation used GM20431 cells.
    • also using with proper controls and pure DNA.
  • procotols from shendure paper, shendure paper transposition
  • using T7-top2 for transposition reactions t7tspn-top2
  • goal is to compare efficiency of DNA accessibility across different cellular concentrations. Also to test efficiency of tagmentation with custom transposon/transposome on pure DNA, and efficiency of amplification from IVT.
    • Using GM12878 cells.
  • Using mouse MEFs to check for possibility of contamination. want to obtain same type of results with custom transposon/IVT amplifiation as obtained in the previous accessibilty assay performed


  • Improvments from last assay
    • DNase used to remove transposons and blocking primer after IVT and fragmentation
    • Use Rnase III for fragmentation. Random nomaer RT has more possible binding locations
    • Cleaning RNA with Zymo RNA cleaning kit


UCSC genome browser mouse hypersensitivity tracts

  • is present on the UCSC Genome Browser on Mouse July 2007 (NCBI37/mm9) Assembly
  • DNaseI Hypersensitivity by Digital DNaseI from ENCODE/University of Washington


  • could be helpful to do comparisons against but a few issues.
    • immortalized cell lines were generated in house by the lab performing the study and are not buyable off of ATCC
    • other cells lines are harvested from mice at 8 weeks of age, such as cells from the cerebellum or B cell lymphocytes.
    • embryonic cells harvested and also had accessibiity performed on them.
  • Thus can make comparisons from my analysis with MEFs but don't know how comparable they will be to UCSC UW DNase I Hypersensitivity tracts.


Why using RNAase III for fragmentation

  • Rnase III creates 5'-PO4 and 3'-OH termini on the fragments
  • however, Cuts ONLY dsRNA (used DNA blocking primer to block that)
  • Preferentially cuts from 5’ and 3’ ends (used blocking primer)
  • Rnase III results in 2 base 3’ overhangs
  • Used in the generation of siRNAs for knockdown
  • Mg++ fragmentation creates 5' OH and 3' PO4 termini, thus have to do end repair.
    • End Repair with Shrink Akaline phosphatase, PNK, Antarctic phosphatase
  • random nonamer is less selective and can prime off of more sequences


buffers compositions

1X T7 buffer:
400 mM Tris Hcl
8  mM MgCl2
2  mM spermidine-Hcl
25 mM NaCl
PH 7.9

1X NEBNext RNase III Reaction Buffer: 
10 mM Tris-HCl 
10 mM Mg(Cl)2 
1 mM DTT 
60 mM NaCl 
pH 8.3 @ 25°C

10X Poly(A) Polymerase buffer:
500 mM Tris-HCl
2.5 M NaCl
100 mM MgCl2
pH 7.9 @ 25°C

MMLV
Invitrogen (though using clontech)
5X First-Strand Buffer
250 mM Tris-HCl (pH 8.3 at room temperature
375 mM KCl
15 mM MgCl2
0.1 M DTT



Before starting protocols

1. Check if have enough reagents etc for the protocol

  • nextera transposomes
  • transposase/transposome
  • IVT reaction mixture
  • cells etc
  • blocking primer, RNase III, PolyA Polymerase


2. Purify GM12878 DNA from Gm12878 cells with qiaquick columns. quanititate DNA and aliquot to a standard concentration of 60 ng or a multiple of that.


Nextera Tagmentation Protocol

1. See step 3 in IVT protocol. Do the cell lysis section for the "nextera tagmentation samples", samples 7,8,9,14,18.


2. Perform tagmentation, with 1:10 diluted nextera enzyme. (1:50 diluted in reaction)

Dilute the enzyme: 1:10     
For each rxn, used mix of: 
 1ul 5x LMW Buffer
 2ul cell lysate
 1ul diluted enzyme
 1ul H2O
----------------------------
5ul total / reaction
55C 10 min


3. Protease digestion of transposition reactions.

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


4. First step of 2-step PCR thermocycling, terminate curves before saturation.

6   ul Tagmentation reaction
25  ul KAPA QPCR mix
1   ul Orange Primer (10uM)
1   ul Blue Primer (10uM)
0.5 ul Bst Pol (5U/ul)
16.5 ul H2O 
----------------
50 uL total

Orange primer    CCTTGCCAGCCCGCTCAG   18nt
Blue primer      CCTCCCTCGCGCCATCAG   18nt 

PCR cycling. 72C for 3 minutes is for gap fill in.

72C 3min -> 95C 30 sec -> (95C 10sec -> 58C 30 sec -> 72 3min) x 25 -> 72C 3min.
Monitor the reactions on a real-time thermal cycler and terminate them before the curves reach saturation.


5. Begin 2-Step AMPure beads purification to remove primers, add index primers, for low input. (with forward read primer and reverse index primers)

a. see 2-step AMpure beads protocol, start from beginning for first purification

b. After cleaning, second PCR reaction to add index primers 
add the following to above dry tube:
25 uL KAPA HF mix
1  uL adaptor1
1  uL of adaptor2 (barcode)
23 uL nuclease free H2O

c. PCR cycling for addition of index primer
5 cycles
72C 3min -> 95C 30 sec -> (95C 10sec -> 60C 30 sec -> 72 1 min) x 5 -> 72C 3min.

d. second phase of AMpure beads 2-step beads purification.

  • can run on gel and check smears to see for library.
  • should be ready for sequencing now?


IVT Protocol

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

1. annealing of ME sequence to T7 transposon sequence

    • a. Make 100 uM stock solution of T7tspn-top2 and T7tspn-bot.
    • b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
    • c. cool to RT at 0.1 C/s


2. transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 20 minutes at RT
1.25 uL of annealed transposon
1.25 uL of 100% sterile glycerol
2.50 uL of Ez-TN5 transposase
  • store at -20, is good for a year


3. Prepare samples, lyse cells with lysis buffer

use pure GM12878 DNA

Samples:
1.  1000 cells IVT
2.  500  cells IVT
3.  100  cells IVT
4.  6   ng purified DNA IVT
5.  600 pg purified DNA IVT
6.  60  pg purified DNA IVT
7.  1000 cells nextera tagmentation
8.  500  cells nextera tagmentation
9.  100  cells nextera tagmentation
10. 6   ng purified DNA nextera tagmentation
11. 600 pg purified DNA nextera tagmentation
12. 60  pg purified DNA nextera tagmentation
13. 1000 cells MEFs IVT
14. 1000 cells MEFs nextera tagmentation
15. 1000 cells lysed without transposome complex IVT
16. pure DNA only (6 ng) IVT
17. Nuclease free H20 only IVT
18. 1000 cells lysed without transposome complex nextera tagmentation
19. pure DNA only (6 ng) nextera tagmentation
20. Nuclease free H20 only nextera tagmentation


  • 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 1000, 500, 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
  • incubate below mixtures at 37C for 30 mins.
' 1. 1K IVT 2. 500 IVT 3. 100 IVT 7. 1K nxta 8. 500 nxta 9. 100 nxta 13. 1K MEF IVT 14. 1K MEF nxta 15. 1K IVT con 17. NTC IVT 18. 1K nxta con 20. NTC nxta
cells 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul PBS 1 ul 1 ul PBS
2X LB 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul


4. transposition reaction, using (T7tspn-top2)

  • add the below into one tube and incubate for 10 minutes at 55C.
1 uL nextera HMW 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)
  • IVT on filled in reaction and not filled in reaction in to show fill-in is working.


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)



8. Clean RNA with with Zymo RNA Clean & Concentrator-5 Kit

  • can quantitate with Qubit if needed


9. Perform RNase III fragmentation (NEB):

Starting Material: Purified mRNA (50–250 nanograms)

 1. Add 5' end blocking DNA primer and do annealing to form DNA-RNA hybrid.
 *a. add 2 uL of blocking oligo (10 uM) to aliquot RNA sample that will be used in step 2.
     use T7-frag-block-top2 for T7-top2.  T7-frag-block for T7-top3
 *b. Incubate at 95C for 2 minutes.
 *c. cool to RT at 0.1 C/s


2. Mix the following components in a sterile PCR tube:

 X  uL Purified mRNA + blocking primer (50-250 nanograms)
 .5  uL RNase III (1 unit/μl)
 1   uL RNase III Reaction Buffer (10X)
 5.5 uL Nuclease-Free Water
 add in DNA primer to protect 5' end since don't want degradation??
 _______
 10 uL total volume


3. Incubate in a preheated thermal cycler for 5 minutes at 37°C.

4. Transfer tube to ice.


10. DNase I digestion and Zymo RNA clean and concentrator cleanup. (removes blocking primer and left over transposon too so wont be later possible PCR issues)

  • Resuspend in appropriate volume in nuclease free H2O.
  • not doing protease digestion since seems it wasnt as effective in previous RNase III fragmentation


11. Quanitate RNA with QUbit


12. Poly(A) Addition with polyA polymerase (Enzymatics)

  • enzymatics PolyA polymerase.
a. assemble reaction:
   2 uL 5X SMART MMLV first strand buffer (rather than 10X polyA buffer)
   1 uL polyA enzyme
   1 uL 10 mM ATP
   bring to 10 uL with RNA or w/e

b. Incubate at 37C for 10 minutes

c. Heat inactivate at 70C for 20 minutes. (rui and NEB)



13. single strand synthesis MMLV RT (Clontech)

20 uL reaction

1. Add 2.5 uL 20 uM primer stock to RNA sample. Bring to final volume of 12.5 uL with
   Nuclease free H2O (one from BENG160 class, T20VN_PE_R)

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 uM DTT
   1  uL N-H20
  .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ____
   20 uL total

4. Incuvate at 42C for 60 minutes

5. Terminate the reaction by heating at 70C for 10 minutes



14. 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 before curves saturate (usually cycle 6-7)


15.qiaquick cleanup

  • can quanitate with nanodrop
  • run qiaquick to clean sample before performing qPCR.


16. Gel Size selection

  • gel size select from 400-800 bp, follow gel size selection protocol
  • do not need to include controls.


17. Cloning and Transformation, then genewiz sequencing for verification of inserts


18. Submit for sequencing if genewiz sequencing checks out.


Results

  • bal hbal bah a



Notes ETC

  • since previous frag block was designed for T7-top3.
T7-frag-block-top2
5'- CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGATCTCCC/3InvdT/  Tm= 81.45