Brandon:LabNotes/Project1/2013-7-31

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IVT Round 9, neural progenitor cells (NPCs), FACS sorted[edit]

  • USE PBS TO RESUSPEND CELLS!!! mistake made with Round 7


  • Blue is using for RNA-seq data, and there is already published CHIP-seq data on the same cell line. Analyses between these datasets and THS-seq generated data can be made.


  • for beads purification, RNA beads will be used. 30 uL of water will be added to the IVT sample, followed by 100 uL of RNA beads. Drying time after the washes will be 3 minutes. Elutions will be saved just in case samples did not bind to the beads.


  • testing on 1000 cell samples currently gives the best data. will collect 1000 cell, 3000 cell and alot of cells (for DNA purification with FACS)


Why using RNAase III for fragmentation

  • Rnase III creates 5'-PO4 and 3'-OH termini on the fragments
  • however, Cuts ONLY dsRNA (used DNA blocking primer to block that)
  • Preferentially cuts from 5’ and 3’ ends (used blocking primer)
  • Rnase III results in 2 base 3’ overhangs
  • Used in the generation of siRNAs for knockdown
  • Mg++ fragmentation creates 5' OH and 3' PO4 termini, thus have to do end repair.
    • End Repair with Shrink Akaline phosphatase, PNK, Antarctic phosphatase
  • random nonamer is less selective and can prime off of more sequences


buffers compositions[edit]

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



Before starting protocols[edit]

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • nextera transposomes
  • transposase/transposome
  • IVT reaction mixture
  • cells etc
  • blocking primer, RNase III, PolyA Polymerase, ATP, taq polymerase, other primers


2. Purify NPC DNA from NPC cells with Zymo DNA clean and concentrator, and make sure to use right buffer concentrations for isolation of genomic DNA. quanititate DNA with nanodrop.



3. samples this time:

Samples:
Index 49, N2 adaptor  1. RNase III, 1000 NPC cells
Index 50, N2 adaptor  2. RNase III, 1000 NPC cells
Index 51, N2 adaptor  3. RNase III, 1000 NPC cells
Index 52, N2 adaptor  4. RNase III, 1000 NPC cells
Index 53, N2 adaptor  5. RNase III, 3000 NPC cells
Index 54, N2 adaptor  6. Rnase III, 6 ng pure NPC DNA control
Index 55, N2 adaptor  7. Rnase III, 6 ng pure Jurkat DNA control
Index 56, N2 adaptor  8. RNase III, 1000 cells lysed without transposome complex IVT
Index 57, N2 adaptor  9. RNase III, Nuclease free H20 only IVT


IVT Protocol[edit]

  • 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

WASHED SAMPLES 2X WITH PBS SINCE SOLUTION HAD 1.0 mM EDTA IN IT. SAMPLES (1,2) WASHED IN PLATE, SAMPLES 3-5 WASHED WHOLE VIAL AND DILUTED THEM DOWN. PURE NPC DNA SAMPLE WAS PURIFIED FROM THE REST OF THE CELLS WITH ZYMO DNA CLEAN AND CONCENTRATOR, NANODROPPED AND INCLUDED ON ASSAY.

Samples:
Index 49, N2 adaptor  1. RNase III, 1000 NPC cells
Index 50, N2 adaptor  2. RNase III, 1000 NPC cells
Index 51, N2 adaptor  3. RNase III, 1000 NPC cells
Index 52, N2 adaptor  4. RNase III, 1000 NPC cells
Index 53, N2 adaptor  5. RNase III, 3000 NPC cells
Index 54, N2 adaptor  6. Rnase III, 6 ng pure NPC DNA control
Index 55, N2 adaptor  7. Rnase III, 6 ng pure Jurkat DNA control
Index 56, N2 adaptor  8. RNase III, 1000 cells lysed without transposome complex IVT
Index 57, N2 adaptor  9. RNase III, Nuclease free H20 only IVT


  • 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, MEFs) that contain 1000, 500, 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


  • incubate below mixtures at 37C for 30 mins.


4. transposition reaction, using (T7tspn-top2)


  • samples 8-9 get H2O instead of transposome
  • add the below into one tube and incubate for 10 minutes at 55C.
1 uL nextera LMW buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
1.2 uL Nuclease free water
.8 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)


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 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


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



8. Clean RNA with AMPURE RNA beads. elute in 10 uL or less.

  • Protocol changes:
    • 1. add 30 uL to IVT reaction to bring to 50 uL total before starting protocol.
    • 2. Use 1.8:1 ratio of beads, thus 80 uL of beads for 50 uL of sample in this case
    • 3. Drying time after the washes is 3 minutes.
  • can quantitate with Qubit or on TBU gel.


9. RNase III fragmentation (NEB), Mg++ fragmentation:


Mg++ fragmentation:

a. RNA fragmentation with MgCl and end repair with T4PNK

 X uL   RNA (100ng-200 ng)
 .5 uL   10x Fragmentation buffer (or dilute to 5X and use 1 uL)
 3-X uL  Nuclease-free H2O
 __________
 5 uL    Total

b. Incubate at 94C for 5 min, place the tube on ice.
   Then perform the end repair as following:

  5 uL    RNA (100 ng total)
  .8 uL  10x Polymerase buffer (PAP buffer)
  .8 uL  10mM ATP
  1.4 uL  T4PNK enzyme
  ____________
  8   uL    Total

  37 C for 30 mins.

c. proceed to PAP addition when ready.


RNase III fragmentation:

use 100-200 ng of RNA
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.
     use T7-frag-block-top2 for T7-top2.  T7-frag-block for T7-top3
 *b. Incubate at RT for 5 minutes (mix well)


2. Mix the following components in a sterile PCR tube:

 a. 5 uL Purified mRNA + blocking primer (50-250 nanograms)
   .5  uL RNase III (1 unit/μl)
   .8  uL RNase III Reaction Buffer (10X)
    X uL Nuclease-Free Water
   add in DNA primer to protect 5' end since don't want degradation??
   ____________
   8.5 uL total volume

 b. Incubate in a preheated thermal cycler for 5 minutes at 37°C.

 c. Heat inactivate RNase III at 65C for 20 minutes.

 d. Immediately cool on ice and proceed to PAP addition.


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

  • enzymatics PolyA polymerase.

ALL RNA SAMPLES

a. to each each add:
   1.2 uL polyA enzyme
   .8 uL 10 mM ATP
   ________
   10.5 uL (should be)
 
b. Incubate at 37C for 10 minutes

c. proceed immediately to Zymo cleaning.


11. Zymo RNA clean and concentrator cleanup.

  • Resuspend in appropriate volume in nuclease free H2O (10 uL last time)


12. 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 for 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



13. second strand synthesis (qPCR) (KAPA), addition of barcodes

Samples:
Index 49, N2 adaptor  1. RNase III, 1000 NPC cells
Index 50, N2 adaptor  2. RNase III, 1000 NPC cells
Index 51, N2 adaptor  3. RNase III, 1000 NPC cells
Index 52, N2 adaptor  4. RNase III, 1000 NPC cells
Index 53, N2 adaptor  5. RNase III, 3000 NPC cells
Index 54, N2 adaptor  6. Rnase III, 6 pure NPC DNA control
Index 55, N2 adaptor  7. RNase III, 1000 cells lysed without transposome complex IVT
Index 56, N2 adaptor  8. RNase III, Nuclease free H20 only IVT


KAPA SYBR FAST qPCR mix until saturation, X35 cycles

12.5 uL KAPA SYBR FAST qPCR mix
2    uL primers, 2 uL F, 2 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX])
.5  uL H2O
10    uL DNA template (use half
RT reaction)
_____________
25 uL

KAPA SYBR cycles:
98C 3min, (98C for 30s, 60C for 30s, 72C for 2 min) X35, 72C for 5 min, 4C forever

  • terminate before curves saturate (usually cycle 6-7)


14.qiaquick cleanup

  • can quanitate with nanodrop


15. Gel Size selection

  • gel size select from 400-800 bp, follow gel size selection protocol
  • do not need to include controls.


16. Cloning and Transformation, then genewiz sequencing for verification of inserts


17. Submit for sequencing if genewiz sequencing checks out.


results[edit]

  • RNA quantification results
' intensity ng/ul '
2612.720078 5
1701.430051 2.5 in 10 uL
Index 49, 1. RNase III, 1000 NPC cells 9239.960275 23.18093033 231.8093033
Index 50, 2. RNase III, 1000 NPC cells 8398.20025 20.87167641 208.7167641
Index 51, 3. RNase III, 1000 NPC cells 9152.810273 22.94184617 229.4184617
Index 52, 4. RNase III, 1000 NPC cells 13081.12039 33.71862963 337.1862963
Index 53, 5. RNase III, 3000 NPC cells 10821.08032 27.51851771 275.1851771
Index 54, 6. Rnase III, 6 ng pure NPC DNA control 50108.68149 135.2986975 1352.986975
Index 55, 7. Rnase III, 6 ng pure Jurkat DNA control 138791.8341 378.5888411 3785.888411
Index 56, 8. RNase III, 1000 cells lysed without transposome complex IVT 2492.030074 4.668903423 46.68903423
Index 57, 9. RNase III, Nuclease free H20 only IVT 1701.540051 2.50030177 25.0030177


  • RNA after ~16.5 hours IVT, run on TBU gel
  • can see that it did not really work. washed cells in plate twice before transposition and probably lost them (samples 1,2). washed the vial of cells twice (samples 3,4,5) and maybe lost them there too. NTCs worked.
  • will not continue experiment.

File:ZhangLab 2 2013-08-01 15hr 31min-labeled.jpg




future and conclusions[edit]

  • Based on positive Jurkat DNA control and NPC pure DNA the assay is working. Most probable is the cells were lost during PBS washes before performing transposition since they were at such small quantity. Washes were performed because the sheath buffer solution contained 1.0 mM Disodium EDTA. Actually 8X concentrate, thus final concentration is .125 mM However upon further examination the amount of EDTA present may not affect transposition.
    • NEB EDTA stop solution for RNA fragmenation has a final concentration of 5 mM EDTA (50 mM in vial)
    • Ambion (Stop Solution - 200 μL of 200 mM EDTA pH 8.0). final EDTA concentration is 20 mM.


  • final concentration of EDTA in transposition reaction would be .025 to .05 mM. Probably will be fine. However, in storage solutions of enzymes concentrations are around that. Also buffer supplied for transposition has 25 mM Mg(OAc)2, which is relatively high.


  • EDTA in enzyme solutions
  • however in these solutions they are usually diluted 5-10X.
0.5 mM EDTA  - NEB Rnase III (storage solution)
0.1 mM EDTA  - MMLV storage soultion
1 mM EDTA    - NEB T7 storage buffer


  • As depicted below, sheath buffer is basically PBS with 1.0 mM Disodium EDTA (at 8X concentration) which is well below that used in final concentrations for RNA fragmentation stop solutions.


  • sheath buffer composition (8X concentration)
  • reconstituted with ddH2O
Biosure Flow Cytometry Sheath solution (CAT# 1027)
1.0 mM Disodium EDTA
1.9 mM Potassium phophate Monobasic (KH2PO4)
3.8 mM Potassium Chloride (KCl)
16.6 mM Sodium phosphate Dibasic (Na2HPO4•2H2O)
139  mM Sodium Chloride  (NaCl)
NONE - Preservative free


  • PBS buffer concentrations (1X concentration)
Salt	Concentration	Concentration
              (mmol/L) (g/L)
NaCl           137     8.01
KCl            2.7     0.20
Na2HPO4•2H2O   10      1.78
KH2PO4         2.0     0.27
pH 7.4


  • also in this exchange, in order to neuatlize EDTA, Mg++ can be added in a 1:1 molar ratio, which can be done experimentally to see if there are any differences.
>Subject: EDTA-Magnesium relationship
>From: Michael Allen (m3allen at sciborg.uwaterloo.ca)
>Date: Thu 30 Mar 2000 - 20:33:02 BST

>I was wondering if any of you could tell me how many divalent cations
>(Mg2+ specifically) a single EDTA (ethylenediaminetetraacetic acid)
>molecule can bind at one time.

>I have searched high and low for this, but could not find a good answer.

>Thanks in advance

>Mike Allen
>University of Waterloo
>Ontario, Canada

Chelation of magnesium by EDTA occurs at a 1:1 molar ratio.  The stability
constants of EDTA for Ca++ and Mg++ are 10.61 and 8.83.  The log10 of the
difference (1.78) is approximately 60, therefore the ratio of complexed
Ca++ to Mg++ in an equimolar mix is about 60:1.  EGTA has a lower stability
constant for Mg++, so the ratio is about 4 e 5.
The Ki at pH 7 or 8 is the "apparent" value at a particular pH which
considers all forms of complexing species, rather than only the most
anionic.  EDTA has 4 pKa values (1.99, 2.67, 6.16 and 10.26) and, at pH
7.0, a = 2.08 e 3, so that log a = 3.3.  For Mg++:EDTA at pH 7.0: log Ki
(apparent) = 8.7 - 3.3 = 5.4.  See R. M. C. Dawson, D. C. Elliot, W. H.
Elliot and K. M. Jones 1969, Data for Biochemical Research.  Oxford Univ.
Press, New York.

www.biotechniques.com/ has a free chelator calculator program available to
subscribers:
 PACKCHEL.ZIP  &nbspSize: 54521 byte(s).
Chelator Calculation of total and free divalent cations obtained using
metal chelators. The program automatically recalculates metal-chelator
stability constants for effects of ionic strength, temperature, and pH. By
editing the
datafile, one may add/delete/change chelators, metals, or pH buffers
present in the original CHELDFLT.DAT datafile or any other datafiles
created with CHELATOR.

Tim Fitzwater
Gilead Sciences

---




  • Lastly, can do an experiment with GM12878 cells where the cells are washed with sheath buffer instead of PBS and quantify RNA after IVT. Samples could be: 1000 cells, 1000 cells extra MgCl2 in 1:1 ratio to neutralize, Jurkat DNA control.
    • This will tell us if the EDTA in sheath buffer is affecting the reaction at all, or if EDTA can be neutralized by addition of MgCl2.
    • However since the given concentration of Mg++ in the transposition reaction is 25 mM, maybe that will easily neutralize it.



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