Brandon:LabNotes/Project1/2013-6-10

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AMPURE RNA beads testing 2, live cells, pure DNA[edit]

  • experiment for AMPURE RNA/DNA beads testing worked here, when using normal ratios of 1 to 1 for beads to sample. not sure why. Samples were then used for downstream processing to see sequencing differences when AMPURE RNA beads, DNA beads, and Zymo kit are used for purification. However during multiple instances, purifying with beads resulted in almost complete sample loss, and need to figure out why.


  • in last experiment with AMPURE RNA beads beads testing, live cells there was much sample loss after purification, but recovery was fine with Zymo RNA purification columns. Now trying experiment again and collecting elutions to see where sample loss is. Will also try experiment with different bead to sample ratios since that may be the issue too. UPDATE 7/2/2013 SAMPLE LOSS WITH CELLS WAS BECAUSE CELL MEDIA WAS USED!! WASH AND RESUSPEND IN PBS!!!


  • if works can use sample for downstream processing. such as the polyA experiment


  • UPDATE for second experiment can bring IVT sample to 50 uL with nuclease free water, then at 2:1 ration of beads (100 uL beads), and follow beads protocol as normal but with short (3 minute) drying time.



Before starting protocols[edit]

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • nextera transposomes
  • transposase/transposome
  • RNA clean and concentrator, AMPURE beads
  • IVT reaction mixture
  • cells etc
  • blocking primer, RNase III, PolyA Polymerase, ATP, taq polymerase, KAPA SYBR FAST, T20VN_PE_R


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 (for IVT:
1. 1K cells cells beads T2
2. 1K cells beads T1
3. 6 ng Pure DNA beads T2
4. 6 ng Pure DNA beads T1
5. NTC (no tspn) beads T2
6. NTC (no tspn) beads T1


Samples for Rnase III titration:

Index 49, N2 adaptor, 1-7. 1X RnIII cells sample (528 ng RNA)
Index 50, N2 adaptor, 2-8. No RnIII (200 ng RNA)
Index 51, N2 adaptor, 3-8. 1XRnase III (0 ng RNA)
Index 52, N2 adaptor, 4-8. 1X RnIII (100 ng RNA)
Index 53, N2 adaptor, 5-8. 1X RnIII (200 ng RNA)
Index 54, N2 adaptor, 6-8. 1X RnIII (200 ng RNA) NO BP
Index 55, N2 adaptor, 7-8. 1X RnIII (400 ng RNA)
Index 56, N2 adaptor, 8-8. 1X RnIII (800 ng RNA)
Index 57, N2 adaptor, 9-8. 1X RnIII NTC sample


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


  • 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.
Samples (for IVT:
1.  1K cells cells beads T2
2.  1K cells beads T1
3.  6 ng Pure DNA beads T2
4.  6 ng Pure DNA beads T1
5.  NTC (no tspn) beads T2
6.  NTC (no tspn) beads T1


' 1. 1K cells cells beads T2 2. 1K cells beads T1 3. 6 ng Pure DNA beads T2 4. 6 ng Pure DNA beads T1 5. NTC (no tspn) beads T2 6. NTC (no tspn) beads T1
cells 1 ul 1 ul 1ul pure 1 ul pure 1ul N-H2O 1 N-H2O
2X LB 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul


4. transposition reaction, using (T7tspn-top2)

  • 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 with AMPURE RNA beads. elute in 10 uL or less.

  • T1 is normal 1:1 ratio and collecting washes/elutions
  • T2 is add 30 uL N-H20 (50 uL total), then add 2 volumes of beads (100 ul)
  • can quantitate with Qubit or on TBU gel.


UPDATE 6/17/2013 PERFORMED RNASEIII FRAGMENTATION ON SAMPLES FROM T2 THAT WORKED. 1K CELLS AND 6 NG PURE DNA. USED PURE DNA SAMPLES TO SEE HOW DIFFERENT RNA CONCENTRATIONS AFFECTS SEQUENCING. ALL 1K CELLS SAMPLE RNA USED IN ONE REACTION SINCE AMOUNTS WERE LOWER. NEW RNASEIII, NO MAGNESIUM FRAGMENTATION

Samples:

' ng/ul intensity total ul ng total for 100 ng/ul for 200 ng/ul for 200 ng/ul for 400 ng/ul for 800 ng/ul
7. 1 uL elute 1. 1K cells T2 52.85895199 18910.36056 10 528.5895199 concentrate to <5 uL
8. 1 uL elute 3. 6 ng pure DNA T2 240.3917933 80809.70241 10 2403.917933 0.415987579 0.831975157 0.831975157 1.663950314 3.327900628

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:

sample RNA RnIII RnIII buffer BP H2O
1-7. 1X RnIII cells sample (528 ng RNA) <5.2 uL 0.5 0.8 2 5.2 - RNA
2-8. No RnIII (200 ng RNA) 0.831975157 0 0.8 2 4.868024843
3-8. 1XRnase III (0 ng RNA) 0 0.5 0.8 2 5.2
4-8. 1X RnIII (100 ng RNA) 0.415987579 0.5 0.8 2 4.784012421
5-8. 1X RnIII (200 ng RNA) 0.831975157 0.5 0.8 2 4.368024843
6-8. 1X RnIII (200 ng RNA) NO BP 0.831975157 0.5 0.8 0 6.368024843
7-8. 1X RnIII (400 ng RNA) 1.663950314 0.5 0.8 2 3.536049686
8-8. 1X RnIII (800 ng RNA) 3.327900628 0.5 0.8 2 1.872099372
9-8. 1X RnIII NTC sample 0 0.5 0.8 2 5.2


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, 1-7. 1X RnIII cells sample (528 ng RNA)
Index 50, N2 adaptor, 2-8. No RnIII (200 ng RNA)
Index 51, N2 adaptor, 3-8. 1XRnase III (0 ng RNA)
Index 52, N2 adaptor, 4-8. 1X RnIII (100 ng RNA)
Index 53, N2 adaptor, 5-8. 1X RnIII (200 ng RNA)
Index 54, N2 adaptor, 6-8. 1X RnIII (200 ng RNA) NO BP
Index 55, N2 adaptor, 7-8. 1X RnIII (400 ng RNA)
Index 56, N2 adaptor, 8-8. 1X RnIII (800 ng RNA)
Index 57, N2 adaptor, 9-8. 1X RnIII NTC sample



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]

  • TBU samples after IVT, and before purification. cell samples have a slightly less smear then usual. Pure DNA samples look normal
' ng/ul intensity '
5 2077.610062
2.5 1089.340032 in 20 uL
1 65.11745272 25842.52077 1302.349054
2 51.66176753 20523.38061 1033.235351
3 165.8200441 65651.06196 3316.400882
4 196.650637 77838.64232 3933.012739




  • For first set of samples, ran AMPURE RNA beads as normal. with 20 uL of IVT reaction, added 20 uL of beads and ran through the procedure.
  • can see that most of the sample is still in the supernatent (flow though), during first step of beads purification. For the pure DNA sample some was eluted in the elution, however most of it is in the flow though.


  • Using first set of trial samples
Samples:
2. 1K cells cells beads T1
4. 6 ng Pure DNA beads T1
6. NTC (no tspn) beads T1
  • TBU on beads washes, elutions etc.
    • 4/40 uL supernatent used
    • 5 uL resuspended beads
    • 1/10 uL elution used

File:ZhangLab 2 2013-06-11 16hr 12min-labeled.jpg



  • For second set of trial samples, first 30 uL of water was added to samples, and then 100 uL of beads for a total of 150 ul in solution. Rest of the beads purification protocol was followed as normal, with ~3 minutes used for drying the beads.
  • here most of the sample is in the elution for both pure DNA and where 1K cells were used. DNA band at ~80 bp is lost, which is what is wanted. The beads still retain some pure DNA sample (lane 5) probably because there is alot of sample. can even try a higher beads ratio.


  • second set of trial samples
Samples:
1. 1K cells beads T2
3. 6 ng Pure DNA beads T2
5. NTC (no tspn) beads T2
  • TBU on beads washes, elutions etc.
    • 4/150 uL supernatent used
    • 5 uL resuspended beads
    • 1/10 uL elution used

File:ZhangLab 2 2013-06-11 19hr 08min-labeled.jpg


  • quantification of results. can see with T1 samples, especially pure DNA sample, most of the sample is in the supernatant. When the altered protocol was used with more beads, most of the sample was in the elution, with ~40-50% sample loss.
' ng/ul intsnity total volume ' ' '
5 2772.840083
2.5 1662.26005 total ng originally in sample loss
1. beads flow through 2. 1K cells T1 9.771560806 4892.520146 38 371.3193106 1302.349054 71.49%
2. beads flow through 4. 6 ng pure DNA T1 105.9414225 47614.25142 38 4025.774055 3316.400882 -21.39%
3. beads flow through 6. NTC (no tspn) T1 -1.067640332 77.40000231 38 0 N/A
4. beads after elution 2. 1K cells T1 2.957868861 1865.660056 5 14.78934431 1302.349054 98.86%
5. beads after elution 4. 6 ng pure DNA T1 9.601221884 4816.850144 5 48.00610942 3316.400882 98.55%
6. beads after elution 6. NTC (no tspn) T1 -1.40309568 -71.62000213 5 0 N/A
7. 1 uL elute 2. 1K cells T1 3.638751823 2168.130065 10 36.38751823 1302.349054 97.21%
8. 1 uL elute 4. 6 ng pure DNA T1 27.48280178 12760.42038 10 274.8280178 3316.400882 91.71%
9. 1 uL elute 6. NTC (no tspn) T1 0.149899152 618.2700184 10 1.498991518 N/A
ng/ul intensity total volume (ul)
5 3113.460093
2.5 2288.280068 total ng originally loss
1. beads flow through 1. 1K cells T2 -3.608182457 272.1400081 150 0 1033.235351 100.00%
2. beads flow through 3. 6 ng pure DNA T2 9.117192613 4472.430133 150 1367.578892 3933.012739 65.23%
3. beads flow through 5. NTC (no tspn) T2 -3.266135873 385.0400115 150 0 N/A
4. beads after elution 1. 1K cells T2 8.192848833 4167.330124 5 40.96424416 1033.235351 96.04%
5. beads after elution 3. 6 ng pure DNA T2 45.27912092 16408.47049 5 226.3956046 3933.012739 94.24%
6. beads after elution 5. NTC (no tspn) T2 -4.005307933 141.0600042 5 0 N/A
7. 1 uL elute 1. 1K cells T2 52.85895199 18910.36056 10 528.5895199 1033.235351 48.84%
8. 1 uL elute 3. 6 ng pure DNA T2 240.3917933 80809.70241 10 2403.917933 3933.012739 38.88%
9. 1 uL elute 5. NTC (no tspn) T2 33.8997552 12652.46038 10 338.997552 N/A



  • UPDATE 6/17/2013 RNA CONCENTRATION TESTING
  • used samples after beads purification. Pure DNA sample #8 was used for different RNA amounts. 1K cells was used as one sample.
  • TBE after barcode addition

File:ZhangLab 2 2013-06-13 19hr 12min-labeled.jpg


  • quantification for gel size selection
' ' ng/ul intensity ' '
5 3323.790099
2.5 1888.290056 for 55 ng sample
49 1-7. 1X RnIII cells sample (528 ng RNA) 13.96048415 8468.900252 3.939691446
50 2-8. No RnIII (200 ng RNA) 12.69292929 7741.070231 4.33312112
51 3-8. 1XRnase III (0 ng RNA) 4.94235458 3290.690098 11.12829909
52 4-8. 1X RnIII (100 ng RNA) 5.597439916 3666.840109 9.825920568
53 5-8. 1X RnIII (200 ng RNA) 7.248066876 4614.630138 7.588230206
54 6-8. 1X RnIII (200 ng RNA) NO BP 7.927220481 5004.600149 6.938119122
55 7-8. 1X RnIII (400 ng RNA) 14.98146987 9055.15027 3.671201856
56 8-8. 1X RnIII (800 ng RNA) 34.5530303 20293.1406 1.591756194
57 9-8. 1X RnIII NTC sample 3.968443051 2731.470081 ---
9 49.01633961 total uL
55 9.169389934 dye to add
495 58.18572954 sum



  • after gel size selection

File:ZhangLab 2 2013-06-17 10hr 00min.jpg



  • validation of gel size selection
  • selected for ~220-800 bp, decent smear.

File:ZhangLab 2 2013-06-17 14hr 19min-labeled.jpg

conclusions[edit]

  • will now add 30 uL of water was added to samples, and then 100 uL of beads for a total of 150 ul in solution. Rest of the beads purification protocol will be followed as normal, with ~3 minutes used for drying the beads.


  • will process this sample through to test replicates, to see if the polyA issue solved.


  • can do one more trial of beads purification, increasing ratio of beads to sample again to ensure everything is working correctly.


  • UPDATE 7/2/2013 SAMPLE LOSS WITH CELLS WAS BECAUSE CELL MEDIA WAS USED!! WASH AND RESUSPEND IN PBS!!! However, RNA concentration testing information worked and is useful.


  • 1X RNase concentation seems to work best for RNA fragmentation. To high and to low and transposase does not work well. Probably because optimal buffer concentrations are maintained


  • AND use more RNA!!! (1 ug sample had best results with 1X Rnase III). Regardless of Rnase III concentration, adding more initial sample works better.


  • NTC has nearly all polyA reads. Was Zymo purified and thus had lots of left over transposon. 10 A's are present in 99.4% of reads. 20 A's in 90.95% of reads. Thus PAP is probably polyA tailing transposon
    • Thus if cannot use polyA or find a way to more efficient will greatly incerase amount of unique reads.