Brandon:LabNotes/Project1/2012-6-29

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Measuring tagmentation efficiency cells versus 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.

If need to make more 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


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)

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. without transposome complex
8. pure DNA only
9. 1000 cells lysed DNA only
10. Nuclease free H20 only
  • add the below into one tube and incubate for 8 minutes at 55C.
1 uL nextera HMW buffer
1 uL genomic DNA (5 ng Jurkat DNA)
2 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


4. 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


5. 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.


6. should now be ready for IVT.

Maxiscript (Ambion) T7 Protocol

DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.

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


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.

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:
1. Add 30 uL water to DNase-I treated transcription reaction to bring the volume to 50 uL.
2. Add 3 volumes 10% EtOH, .1 volumes 3M NaOAc, and 1/300 volumes glycol Blue to the solution obtained.
   In this case:
   5   uL NaOAc (mix after adding)
   150 uL  100% EtOH
   1   uL (1/10 dilution Glycol Blue)

3. Chill solution in -80 for 30 minutes, cool centrifuge to 4C

4. Spin at 4C for >15 minutes at max speed. Chill 75% EtOH. 

5. Blue pellet should be visible, Discard supernatent.

6. Wash the pellet with cold 75% EtOH

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.

8. Store RNA at -80, in aliquots if desired. Aliquot in use can be at -20.

   
  • Now can nanodrop for quanitation or run gel. fragment RNA, polyA tail addition, adapter addition to 5' and 3' ends.