Brandon:LabNotes/Project1/2013-1-17

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IVT round 4[edit]

  • doing IVT protocol again to show results are reproducible, and testing changes made to protocol after experiments.


  • troubleshooted why IVT was not working before and found RNase III was bad. got new RNase III.
  • did testing post RNA fragmentation processing methods and found one that has higher efficiency and will use that
  • testing on 1000 cell samples since that gives the best data
  • will try out magnesium fragmentation of RNA and RNase III fragmentation to do a better comparison between samples. will also try incubation with transposome at 37C for 30 minutes opposed to heating for 55C for 10 minutes. heating to 55C could disrupt proteins and chromatin structure.


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 GM12878 DNA from GM12878 cells with DNeasy blood and tissue kit for cells (USE PROTEINASE K (invitrogen) AND protease (qiagen) AND RNAase A). quanititate DNA with nanodrop.

GM12878 DNA purification method used (followed DNeasy kit protocol for washes etc):

a. pellet cells

b. resuspend in:
    1. 100 ul PBS
    2. 10 uL proteinase K
    3. 3 uL 5 AU Qiagen protease
    4. 4 uL A797A promega RNase solution (RNase A)

c. add 200 uL buffer AL, incuabate at 56C for 10 minutes

d. step 3 in DNeasy kit protocol until finish


3. samples this time:

Samples:
Index 49, N2 adaptor  1.  RNase III, 1000 cells IVT
Index 50, N2 adaptor  2.  RNase III, 1000 cells IVT Zymo <200 bp RNA removal
Index 51, N2 adaptor  3.  RNase III, 1000 cells IVT 37C, 30 min tagmentation
Index 52, N2 adaptor  4.  RNase III, 1000 cells IVT 3X [NT]
Index 53, N2 adaptor  5.  RNase III, 1000 cells lysed without transposome complex IVT
Index 54, N2 adaptor  6.  RNase III, Nuclease free H20 only IVT
Index 55, N2 adaptor  7.  Mg++, 1000 cells IVT
Index 56, N2 adaptor  8.  Mg++, 1000 cells IVT Zymo <200 bp RNA removal
Index 57, N2 adaptor  9.  Mg++, 1000 cells IVT 37C, 30 min tagmentation
Index 58, N2 adaptor  10. Mg++, 1000 cells IVT 3X [NT]
Index 59, N2 adaptor  11. Mg++, 1000 cells lysed without transposome complex IVT
Index 60, N2 adaptor  12. Mg++, 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

Samples:
1. 1000 cells IVT
2. 1000 cells IVT Zymo <200 bp RNA removal)
3. 1000 cells IVT 37C, 30 min tagmentation
4. 1000 cells IVT 3X [NT]
5. 1000 cells lysed without transposome complex IVT
6. 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


  • make each cellular concentration in triplicate for AluI amplification with PCR for cell number quantification! store samples and perform quantification later. Use pure DNA nanodropped controls!!
AluI PCR quant
5 uL AluI 245-263 primer
5 uL AluI 21-40 primer
X sample 
X water
_________
25 uL
  • incubate below mixtures at 37C for 30 mins.
' 1. 1K IVT 2. 1K IVT 3. 1K IVT, 37C 30min 4. 1K IVT 2X [NT] 5. 1K lysed 6. N-H20/PBS con
cells 1 ul 1 ul 1 ul 1 ul 1 ul 1 PBS
2X LB 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul


4. transposition reaction, using (T7tspn-top2)

samples 1,2,4,5,6 do 55C 10min sample 3 do 37C, 30 min

  • 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

  • !!!!! sample 4 used 3 uL of each NT!!!!!!!
  • 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
  !!!!! sample 4 used 3 uL of each NT!!!!!!!


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



8. Clean RNA with with Zymo RNA Clean & Concentrator-5 Kit. elute in 10 uL or less.

  • 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 cells IVT
Index 50, N2 adaptor  2.  RNase III, 1000 cells IVT Zymo <200 bp RNA removal
Index 51, N2 adaptor  3.  RNase III, 1000 cells IVT 37C, 30 min tagmentation
Index 52, N2 adaptor  4.  RNase III, 1000 cells IVT 3X [NT]
Index 53, N2 adaptor  5.  RNase III, 1000 cells lysed without transposome complex IVT
Index 54, N2 adaptor  6.  RNase III, Nuclease free H20 only IVT
Index 55, N2 adaptor  7.  Mg++, 1000 cells IVT
Index 56, N2 adaptor  8.  Mg++, 1000 cells IVT Zymo <200 bp RNA removal
Index 57, N2 adaptor  9.  Mg++, 1000 cells IVT 37C, 30 min tagmentation
Index 58, N2 adaptor  10. Mg++, 1000 cells IVT 3X [NT]
Index 59, N2 adaptor  11. Mg++, 1000 cells lysed without transposome complex IVT
Index 60, N2 adaptor  12. Mg++, 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 whole 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
' 5 2577.530077 ' '
2.5 1737.350052
ng/ul 218.1504559 74211.43221 sample 1
ng/ul 207.2097348 70534.5621 sample 2
ng/ul 158.1947023 54061.98161 sample 3
ng/ul 273.4311993 92789.74277 sample 4
smpl in 10 uL smpl using 200 ng each sample
1 2181.504559 ng RNA total 1 0.916798451
2 2072.097348 ng RNA total 2 0.965205617
3 1581.947023 ng RNA total 3 1.26426484
4 2734.311993 ng RNA total 4 0.731445426


  • RNA after ~16.5 hours IVT, run on TBU gel

File:ZhangLab 2 2013-01-18 11hr 32min-labeled.jpg


  • barcode addition PCR curves. terminated at end of 5th cycle since curves that went up were saturated. still don't know why some work and some don't.
  • Curves are in order of sample listings below.
Samples:
Index 49, N2 adaptor  1.  RNase III, 1000 cells IVT
Index 50, N2 adaptor  2.  RNase III, 1000 cells IVT Zymo <200 bp RNA removal
Index 51, N2 adaptor  3.  RNase III, 1000 cells IVT 37C, 30 min tagmentation
Index 52, N2 adaptor  4.  RNase III, 1000 cells IVT 3X [NT]
Index 53, N2 adaptor  5.  RNase III, 1000 cells lysed without transposome complex IVT
Index 54, N2 adaptor  6.  RNase III, Nuclease free H20 only IVT


  • RNase III treated samples:

File:2013-01-22 round 4 IVT barcode addition rnase iii.bmp


Samples:
Index 55, N2 adaptor  7.  Mg++, 1000 cells IVT
Index 56, N2 adaptor  8.  Mg++, 1000 cells IVT Zymo <200 bp RNA removal
Index 57, N2 adaptor  9.  Mg++, 1000 cells IVT 37C, 30 min tagmentation
Index 58, N2 adaptor  10. Mg++, 1000 cells IVT 3X [NT]
Index 59, N2 adaptor  11. Mg++, 1000 cells lysed without transposome complex IVT
Index 60, N2 adaptor  12. Mg++, Nuclease free H20 only IVT
  • Mg++ treated samples:

File:2013-01-22 round 4 IVT barcode addition mg++.bmp



  • TBE gel after barcode addition
  • produced good smears, not sure why some are stronger/less stronger

File:ZhangLab 2 2013-01-22 18hr 14min-labeled.jpg



  • amounts used for gel size selection:
' ' Volume ' ' '
5 2590.410077 800 ng total
smpl 2.5 1406.460042 smpl 100 ng/sample (uL needed)
1 45.99499557 22004.82066 1 2.174149574
2 31.80567169 15285.04046 2 3.144093323
3 20.48490223 9923.750296 3 4.881643996
4 15.32247983 7478.930223 4 6.526358728
7 38.87028591 18630.70056 7 2.572659235
8 32.33812661 15537.20046 8 3.092325081
9 36.1191351 17327.81052 9 2.768615575
10 36.11263145 17324.73052 10 2.769114185



  • after gel size selection

File:ZhangLab 2 2013-01-23 17hr 10min-labeled.jpg


data analysis UPDATE 2013/02/08

  • Index 55 is missing. not sure why, possible to have used Indx 58 opposed to 55 since Indx 58 has alot of reads.


  • Rnase III gives lower amount of clonal reads and higher single alignments, while Mg++ gives less percentage wise. However Mg++ over gives more reads and thus total unique reads obtained are about the same.


  • merged files to do an analysis:
  • reslts are satisfactory so far, in terms of peaks called and genomve coverage with coverageBed
  • Rnase III seems better than Mg++ fragmentation, need more data to draw better conclusions
' total unique reads peaks UCSC_overlap_BP total_BP %age
merged_RnIII 11,512,998 14,539 1,398,227 1,733,628 80.65%
merged_Mg++ 7,716,720 10,355 555,604 956,666 58.08%
merged+RnIII_and_mg++ 19,042,895 20,982 2,164,054 2,747,638 78.76%


  • coverage with coveragebed


  • overlap on chr8:70,601,914-70,603,038
  • Rnase III fragmentation seems to have better capture of fragments for peak generation. Even with sample 58, which has a million more reads than any of the RNase III samples
  • Indx49-52 = RNase III
  • Indx56-58 = Mg++
  • last one is Mg++ and Rnase III merged

File:UCSC Mg++ versus RnIII.png


UPDATE 2/26/2013 sequencing status

  • Alan tried doubling library size on sequencer twice but failed and gave very few reads. combination with the previous successful run seems to decrease the quality of the combined data. Thus will not include those sequencing results in analysis


  • Ran out of sample because of those failed sequencing runs, so did gel size selection again on the samples. did not include sample 7 (index 55) since has barcdoe addition was with nextera primer instead of N2 Illuminia one.
' ' Volume ' ' '
5 2590.410077 800 ng total
smpl 2.5 1406.460042 smpl 100 ng/sample (uL needed)
1 45.99499557 22004.82066 1 2.174149574
2 31.80567169 15285.04046 2 3.144093323
3 20.48490223 9923.750296 3 4.881643996
4 15.32247983 7478.930223 4 6.526358728
7 38.87028591 18630.70056 7 2.572659235
8 32.33812661 15537.20046 8 3.092325081
9 36.1191351 17327.81052 9 2.768615575
10 36.11263145 17324.73052 10 2.769114185


  • gel size selection

File:ZhangLab 2 2013-02-25 15hr 41min-labeled.jpg


  • validation of insert size after gel size selection:

File:ZhangLab 2 2013-02-25 19hr 22min-labeled.jpg




UPDATE 3/22/2013

  • ran on one lane of hi-seq at BIOGEM.
  • gave different results for base composition and quality scores than usual libraries. For base composition, there are many more A's per cycle then other nucleotides. This is assumed to be because of the polyA issue where the read1 sequence is polyA'ed. Read qualitites drop off after 20 BP, but they are still phred scores above 30 which is high. maybe because of PCR


  • base composition

File:Sos base lane1.PNG



future and conclusions[edit]

  • blah balh

Blah