Brandon:LabNotes/Project1/2014-4-30

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Testing pooling and purification with AMPURE beads

  • In Testing pooling and purification with AMPURE beads it was found to contain the most IVT product after amplification when compared to EtOH purification or column purification. Also it even though there was only very little amplification from IVT from pooling it still gave IVT product when the transposome reaction buffer was not optimized, while purification with other methods did not.


  • Will try BOTH tagmentation, protease digestion, taq2X fill in and then AMPURE beads purification, AND tagmentation, AMPURE beads purification, and taq2X fill in, to see if there are any IVT amplification efficiency differences. Optimal is taq2x fill in after AMPURE beads so tagemtantion reaction volumes can remain smaller.


  • will dilute transposase 2X to save transposase.


  • Can also test:
1. combining individually barcoded samples and running a large IVT reaction
2. using guandine HCL for proteins denatureation then purification



Before starting protocols

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • sc1 transposomes
  • custom 3' transpson
  • IVT reagents
  • cells etc
  • 5X, 2X taq polymerase
  • Zymo DNA clean and concentrator


  • 2. Samples
1. sc1-T7iBR-Idx25 - 100 cells
2. sc1-T7iBR-Idx26 - 100 cells
3. sc1-T7iBR-Idx27 - 100 cells
4. sc1-T7iBR-Idx28 - 100 cells
5. sc1-T7iBR-Idx29 - 100 cells
6. sc1-T7iBR-Idx30 - 1000 cells
7. sc1-T7iBR-Idx31 - 3 ng pure DNA
8. sc1-T7iBR-Idx32 - NTC

IVT Protocol

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

MAKE 2X DILUTED TRANSPOSASE 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 1.0 uL of DILUTED Ez-TN5 transposase to well.
  • store at -20, is good for a year


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

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 XX cells/uL
  • 3. Count cells again, aliquot cells to sample tubes

CELL LYSIS

  • 1. dilute Lysis Buffer (LB) to 2X, or as needed (from 10X stock)
  • 3. add LB 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

Add all components and incubate at 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)
___________
5 uL total solution


5. Protease digestion of transposase, protease inactivation

To each tube, add:
1 uL  Qiagen Protease, use 1 uL of 11.1 ug/uL (.5 AU/ml) protease (dilute 5.5 uL 20 ug/ul with 4.5 uL N-H2O)
Incubate: 50C 10 minutes, 70C 20 minutes


6. Fill in reaction

  • Add 1 uL of 5X taq polymerase or 6 uL of taq2X. Run at 72C for 3 minutes. (same as nextera)


samples 1-5 pooled, samples 6,7,8 kept separate

Using AMPURE RNA beads since they are verified as RNase etc free


7. Pool samples. Clean with RNA AMPURE beads

  a. pool samples. add 1.5X volumes of N-H2O to each sample
       With 12 uL sample volume:
       i. add 18 uL N-H2O
       ii. add 54 uL beads (1.8X)
  b. follow AMPURE beads cleaning protocol (one wash, 2 EtOH washes)
  c. resuspend in 12 uL H2O
  d. proceed to IVT


8. 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)
  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)



9. Clean with Zymo clean and concentrator

  • 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

  • TBU gel after IVT