Brandon:LabNotes/Project1/2014-6-4

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Guanidine HCl after transposition, or other steps. taq2X on beads, IVT and AMPURE beads[edit]

  • previously AMPURE beads have not really worked, except on occasion, and never work with large cell samples. Will try using ampure beads with guanidine HCl. taq2x fill in and IVT will be done on beads.



  • here EtOH and zymo purification did not work, however maybe it was because a 5X dilution of transposase was done. EtOH pufication with pure DNA has higher RNA yield thus so far AMPURE >= EtOH > Zymo columns

Pooling and purification with Zymo column purification and EtOH purification


  • when searching "guanidine hcl and ampure beads"
  • "Use of SPRI instead of spin filter for tagmentation cleanup. To decrease manual intervention, a magnetic bead‐based method for purifying the tagmentation reactions was implemented. In order to dissociate the tagmentase enzyme from the DNA, guanidine HCl is added to a net concentration of 4 M. Two sequential additions of AMPure bead reagent are effective at purifying the DNA from the rest of the tagmentation reaction components, as shown in Figure 3." http://www.perkinelmer.com/pdfs/downloads/GDE_Nextura_JANUS_NGS_EXpress.pdf
    • This method has already been validated on an automated system, and should work for us.
    • They also perform 2 step beads purification, and that will be tried too.
Add .8X uL beads to sample
Inucubate for 10 minutes
Incubate on magnet for 5 minutes
remove supernatent
Add 1.8X uL beads (1.8X the original volume)
Proceed with incubation, magnet and EtOH washing


Before starting protocols[edit]

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
samples
Transposase is undiluted. 1X concentrated
1. sc1-T7iBR-Idx25, tagmentation, taq2x fill in (no protease), Guanidine HCl, IVT
2. sc1-T7iBR-Idx25, tagmentation, Guanidine HCl, beads pur., taq2x, IVT
3. sc1-T7iBR-Idx25, tagmentation, Guanidine HCl, double beads pur. .8X then 1.8X, taq2X, IVT
4. sc1-T7iBR-Idx26, tagmentation, protease, taq2x, Guanidine HCl, beads, IVT
5. sc1-T7iBR-Idx26, tagmentation, protease, taq2x, IVT (normal protocol control)
6. sc1-T7iBR-Idx26, tagmentation, Guanidine HCl, beads pur., taq2x, IVT (3 ng Jurkat pure dna control)
7. sc1-T7iBR-Idx27, tagmentation, Guanidine HCl, beads pur., taq2x, IVT NTC control



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 1.0 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

1.0 uL 5X Custom Tagmentation buffer
2.0 uL lysed cells/pure genomic DNA
1.0 uL (or .6 uL undiluted) prepared T7 transposomes (MAKE SURE TO ADD LAST) (5X diluted)
___________
4.0 uL total solution


5. Perform either: taq2x, Guanidine HCl, or protease. For guanidine HCl, bring to 15 uL with N-H2O. Add 15 uL 8M guanidine HCl for a total of 30 uL.

    • If using beads next add 1.8X (54 uL) beads or for sample 3., 24 uL beads, then 54 uL beads


ELUTE IN 9.6 or 12 uL DEPENDING, leave beads in solution


6. Fill in reaction

  • Add 2.4 uL of 5X taq polymerase or 3.0 uL of taq2X. Run at 72C for 3 minutes. (same as nextera)


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[edit]

  • total RNA. Lower then usual, but so is control. might be the gel.
' ng/ul intensity '
5 2285.650068
2.5 1316.500039 ng in 10 uL
1. sc1-T7iBR-Idx25, tagmentation, taq2x fill in (no protease), Guanidine HCl, IVT -0.896017128 0 0
2. sc1-T7iBR-Idx25, tagmentation, Guanidine HCl, beads pur., taq2x, IVT 19.80537069 8025.100239 198.0537069
3. sc1-T7iBR-Idx25, tagmentation, Guanidine HCl, double beads pur. .8X then 1.8X, taq2X, IVT 20.47208894 8283.560247 204.7208894
4. sc1-T7iBR-Idx26, tagmentation, protease, taq2x, Guanidine HCl, beads, IVT 5.131455399 2336.61007 51.31455399
5. sc1-T7iBR-Idx26, tagmentation, protease, taq2x, IVT (normal protocol control) 4.44618996 2070.960062 44.4618996
6. sc1-T7iBR-Idx26, tagmentation, Guanidine HCl, beads pur., taq2x, IVT (3 ng Jurkat pure dna control) 101.4021823 39656.92118 1014.021823
7. sc1-T7iBR-Idx27, tagmentation, Guanidine HCl, beads pur., taq2x, IVT NTC control #VALUE! #VALUE!
  • TBU gel after IVT, Guanidine HCl to remove proteins worked

File:ZhangLab 2 2014-06-05 16hr 42min-labeled.jpg


conclusions[edit]

  • Overall will repeat to see if RNA yields increase. Will also try EtOH preciptation again with Guanidine HCl to denature proteins before purification.


  • When pooling will now try using guanidine HCl to remove proteins. Previously 1000 cell samples were not working, maybe since proteins are still bound and/or are not removed efficiently by AMPURe beads alone.


  • Total amout of RNA generated is lower, however it could be the gel since the positive control is lower


  • Since normal process did not work, it could be the protease being added is not working. can try ordering a new protease or using a different one to use the normal protocol without cleaning.