Brandon:LabNotes/Project1/2014-2-24

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Continuation of validation of sc1 transposons[edit]

  • Successfully validated transposons here, however 38,44,59 did not work. Thus going to validate those ones. Will start from the initial step of annealing, will not dilute transpoase, will use 3 ng of Jurkat DNA and will use 1/2 of IVT reagents.


  • datasheet with primer information/concentration/Tm's/etc. File:Fung 020614.xls
  • Bold Transposons generate sufficient IVT product
' 1 2 3 4 5 6
A sc1-T7iBR-Idx25 sc1-T7iBR-Idx33 sc1-T7iBR-Idx41 sc1-T7iBR-Idx49 sc1-T7iBR-Idx57 sc1-T7iBR-Idx65
B sc1-T7iBR-Idx26 sc1-T7iBR-Idx34 sc1-T7iBR-Idx42 sc1-T7iBR-Idx50 sc1-T7iBR-Idx58 sc1-T7iBR-Idx66
C sc1-T7iBR-Idx27 sc1-T7iBR-Idx35 sc1-T7iBR-Idx43 sc1-T7iBR-Idx51 sc1-T7iBR-Idx59 sc1-T7iBR-Idx67
D sc1-T7iBR-Idx28 sc1-T7iBR-Idx36 sc1-T7iBR-Idx44 sc1-T7iBR-Idx52 sc1-T7iBR-Idx60 sc1-T7iBR-Idx68
E sc1-T7iBR-Idx29 sc1-T7iBR-Idx37 sc1-T7iBR-Idx45 sc1-T7iBR-Idx53 sc1-T7iBR-Idx61 sc1-T7iBR-Idx69
F sc1-T7iBR-Idx30 sc1-T7iBR-Idx38 sc1-T7iBR-Idx46 sc1-T7iBR-Idx54 sc1-T7iBR-Idx62 sc1-T7iBR-Idx70
G sc1-T7iBR-Idx31 sc1-T7iBR-Idx39 sc1-T7iBR-Idx47 sc1-T7iBR-Idx55 sc1-T7iBR-Idx63 sc1-T7iBR-Idx71
H sc1-T7iBR-Idx32 sc1-T7iBR-Idx40 sc1-T7iBR-Idx48 sc1-T7iBR-Idx56 sc1-T7iBR-Idx64 sc1-T7iBR-Idx72


  • Will use our custom 5X Transposition buffer from Jeff and Andrew. Tested previously custom 5X tagmentation buffer and was found to be compreable to Nextera LMW, using the end point of IVT product.


  • Differences from normal assay conditions:
    • Reactions carried out at 55C
    • Taq5X will be used for end fill in
    • IVT reagent volumes will also be halved. This will result in a ~10 uL reaction for IVT. determined here sc1 validation changes


Before starting protocols[edit]

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • nextera transposomes
  • custom 3' transpson
  • transposase/transposome
  • IVT reaction mixture
  • cells etc
  • RNase III, PolyA Polymerase, ATP, 5X and 2X taq polymerase, other primers


2. make pure DNA controls for the samples if needed.


3. samples this time:

  • sample list
1. sc1-T7iBR-Idx38
2. sc1-T7iBR-Idx44
3. sc1-T7iBR-Idx59
4. NTC


IVT Protocol[edit]

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

Generation of sc1-T7iBR-IdxXX transposomes

1. annealing of ME sequence to T7 transposon sequence

    • a. Make 100 uM stock solution of sc1-T7iBR-IdxXX and sc1-T7tspn-NoP-bot.
    • b. Incubate 2.5 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 1 minutes. Oligo's now at 10 uM in 25 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. Add 0.5 uL of 100% sterile glycerol to tube
2. Add 0.5 uL of annealed transposon to the 100% sterile glycerol and mix well
4. Add 1 uL of Ez-TN5 transposase to well.
  • store at -20, is good for a year


NOT DOING THIS EXPERIMENT Generation of custom barcode (3' side of fragments)illuminia adaptor transposome

1. annealing of ME sequence to Nextera transposon sequence

    • a. Make 100 uM stock solution of (sc1-T7tspn-NoP-bot, "pMENTS") and (sc-ILA-adaptor).
    • b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 1 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

USING PURE JURKAT DNA, 3 NG

Sample list
1. XX Pure Jurkat DNA for all


CELL WASHING - (wash cells with PBS)

  • 1. count cells, spin down all cells at 250-500 g for 5 minutes
  • 2. wash with 1X PBS, centrifuge 250-500 g for 5 minutes, resuspend to XXXX cells/uL
  • 3. Aliquot cells to sample tubes

CELL LYSIS

  • 1. dilute LB to 2X, or as needed (from 10X stock)
  • 2. refrigerate or warm LB or keep at RT
  • 3. add lysis buffer to cell aliquots, mix briefly (mineral oil optional)
  • 4. transposition ready


LYSIS BUFFER NOTES

  • Lysis buffer is: 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.



4. transposition reaction, using (all sc1-T7iBR-IdxXX's)

make mastermix of 5X tagmentation buffer, 2X lysis buffer, pure DNA, Nuclease free H20

  • add the below into one tube and incubate for 10 minutes at 55C.
  • OR 37C FOR 30 MINUTES
1 uL 5X Custom Tagmentation buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
1 uL Nuclease free water
1 uL (or .6 uL undiluted) prepared T7 transposomes (MAKE SURE TO ADD LAST) (shendure used .625)
___________
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, 20 ug/ul solution add 5.55 uL protease with 4.5 uL N-H20 for 11.1 ug/ul or .5 mAU/ul
Incubate: 50C 10 minutes, 70C 20 minutes


6. Fill in reaction

  • Add 1 uL 5X taq polymerase, run at 72C for 3 minutes. (OR 6 uL taq2X) (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 10 uL with Nuclease free water
  X   uL   DNA template (list 1 ug)
  1   uL   10X Transcription Buffer
 .5   uL   10 mM ATP
 .5   uL   10 mM CTP
 .5   uL   10 mM GTP
 .5   uL   10 mM UTP
  1   uL   T7 Enzyme Mix


b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)



8. Clean with Zymo clean and concentrator now

  • elute samples in 10 uL of N-H2O
  • quanitate with Qubit or on TBU gel.


AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO

Results[edit]

SEE Transposome results for updated table of validated transpons

  • RNA after IVT NOT CLEANED W/ ZYMO YET
  • used undiluted transposase for reactions.
  • used 2 uL for gels
  • 38 still appears light. might need to be purified or reordered.

File:ZhangLab 2 2014-02-25 15hr 47min-labeled.jpg