Brandon:LabNotes/Project1/2012-8-6

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Custom transposon with IVT amplification, round 2

  • testing for stuff
  • list samples


UCSC genome browser mouse hypersensitivity tracts

Why using RNAase III for fragmentation


buffers

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
  • 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.


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

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. 1000 cells without transposome complex
8. pure DNA only (6 ng)
9. 1000 cells lysed DNA only
10. Nuclease free H20 only (cells)
11. Nuclease free H20 only  (pure DNA)


  • 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 10000, 1000, 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


' 4. 10000 5. 1000 6. 100 7. 1000C 9. 1000C 10. NTC 11. NTC
cells 1ul 1ul 1ul 1ul 1ul 1ul PBS 1ul PBS
2X LB 1ul 1ul 1ul 1ul 1ul 1ul 1ul
  • incubate at 37C for 30 mins.


4. transposition reaction, using (T7tspn-top2)

  • add the below into one tube and incubate for 8 minutes at 55C.
1 uL nextera HMW buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
1 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


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.



Rnase III protocol

1. Retrieve IVT RNA.

  • samples:
1. with Blocking Primer, protease treated
2. with blocking primer, EtOH cleaned
3. NTC with blocking primer, protease
4. without blocking primer, protease treated
5. without blocking primer, EtOH cleaned
6. NTC without blocking primer, protease treated

used 5 uL RNA for each sample


2. Perform EtOH cleanup of RNA. (can do columns)


3. 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.
 *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.


4. DNase digestion and Zymo RNA clean and concentrator cleanup. (removes blocking primer 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


4.5 Quanitate RNA with QUbit


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

  • enzymatics PolyA polymerase.
1. 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

2. Incubate at 37C for 10 minutes

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



6. 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



7. 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 curves saturate


8.qiaquick cleanup

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


Results

  • TBU gel after RNase III fragmentat



Notes ETC

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