Matt:LabNotes/2014-3-10

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Agi26k0gap Secondary Rolonies Mattek Dish[edit]

  • Since we now have a successful procedure for generating Secondary Rolonies
  • Since we have optimized PGP1f sample prep by culturing without EGF (Hosuk:LabNotes/2014-3-3)
    • In Harvard Science paper (Lee, J. et al), this culture condition resulted in more mRNA and less rRNA
    • Also microarray data on GEO shows Agi26k0gap probe set targets many high expression transcripts in PGP1f cells Matt:LabNotes/2014-3-7
  • Try to generate highly multiplexed 2nd rolonies with 12k padlock probeset
    • 30.3 pmole of Agi26k0gap prepared LabNotes/2014-3-5
      • 30.3 pmole / 200ul = 0.1515 pmole/ul = 0.1515 umole/L = 151.5nM
        • 151.5nM/12,000 probes = 0.0126 nM = 12.6 pM

Procedure[edit]

Padlock Probe Capture[edit]

  1. Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min
  2. Wash with 1X PBS twice
  3. Anneal Padlock Probes @45C for 28hrs (Put in at 11:30a)
    • 1X Ampligase Buffer
    • ~12.6nM of each padlock probe
    • 0.5 U/ul Ampligase
    • Prepared everything on ice and then pre-heated to 75C for 5 min before adding
Component Volume
10X Ampligase Buffer 20
30.3 pmole Agi26k0gap 58
5U/ul Ampligase 20
H2O 102
Total 200

Secondary RCA[edit]

  1. Aspirate but don't wash
  2. Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
    1. 1ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of 2xSSC/30% formamide
    2. Preheat primer at 60C (Tm = 55C when not in 30% formamide)
    3. Add 200ul and incubate at 45C for 15min.
    4. Aspirate, and wash using 2x SSC twice
    5. Aspirate, and wash using 0.1x SSC twice
  3. Prepare the rolling circle amplification reaction mixtures on ice.
    1. 171 ul ddH2O,
    2. 20 ul 10x Phi29 buffer,
    3. 2 ul 25 mM dNTP,
    4. 4 ul 2 mM aminoallyl dUTP
    5. 3 ul Phi29 DNA polymerase (Epicentre, low concentration)
      • 200ul Total Volume
  4. Add and incubate 20hrs at 30C. (Put in at 4:15pm 3/11/14)
  5. Wash 1X PBS once
  6. Add 200ul BS(PEG)9 mix to each well and incubate @RT for 1hr
  7. Wash 1X PBS twice
  8. Add 200ul 1M Tris pH 8.0 @RT for 30min
  9. Wash 1X PBS twice

Detection[edit]

Initial Imaging[edit]

  • 3/12/2014
  • Check to see if 2nd rolony generation was successful by detecting a large fraction
    • Use 1uM dcProbe0-Cy3 and 1uM dcProbe0-FAM in 30% formamide + 2X SSC
      • This should detect ~1/2 of all 2nd rolonies

Results[edit]

Initial Imaging[edit]

  • Using PISA7 Paramters: Gaussian std (3), upper bound (-2e-4), area upper (50), area lower (4), axratio lower (.6), circ upper (1.6), circ lower (.8), perim conn (8), bkgmult lower (variable)
  • First used PISA to examine the images with varying bkgmult lower (range: 2-9)
' Bkgmult Rolony Count Notes
NoDye_G680_Cy3 7 116
NoDye_G680_Cy3 9 31
NoDye_L2%_G680_FAM 7 202 picked up mostly nuclei
NoDye_L2%_G680_FAM 9 7
dcProbe0-Cy3_G550 7 178
dcProbe0-Cy3_G550 9 162
dcProbe0-Cy3_G550 5 208
dcProbe0-Cy3_G550 3 265 decreasing bkgmult detects more "true" features without significantly counting artifacts
dcProbe0-Cy3_G550 2 278 doesn't detect anymore "true" features, just adds noise
dcProbe0-Cy3_G680 7 544
dcProbe0-Cy3_G680 9 489
dcProbe0-FAM_L5%_G550 7 129
dcProbe0-FAM_L5%_G550 3 209
dcProbe0-FAM_L5%_G550 2 245 still misses many while counting artifacts
dcProbe0-FAM_G550 7 52
dcProbe0-FAM_G550 5 68
dcProbe0-FAM_G550 3 108 even lowering it to 3 still won't count obvious features
dcProbe0-FAM_G600 7 313
dcProbe0-FAM_G600 5 364 good
dcProbe0-FAM_G680 7 519
dcProbe0-FAM_G680 9 375 not bad, pretty similar to G600 bkgmult5

Count Cells in Field of View

  • NoDye_L2%_G680_FAM had nuclei with high intensity fluorescence relative to background, which was really obvious after processing in PISA so counted nuclei in that image
    • 110 Nuclei/Cells counted

File:Agi26k0gap PGP1f InitialDetect NucleiCount110.png

Best Images and PISA Settings

' Image Name Image Feature Count
Cy3 Background NoDye_G680_Cy3_Bkgmult9 File:NoDye G680 Cy3 ch00 PISA7Overlay Bkgmult9.png 31
FAM Background NoDye_L2%_G680_FAM_Bkgmult9 File:NoDye L2% G680 FAM ch00 PISA7Overlay Bkgmult9.png 7
dcProbe0-Cy3 dcProbe0Cy3_G550_Bkgmult3 File:DcProbe0Cy3 G550 Cy3 ch00 PISA7Overlay Bkgmult3.png 265
dcProbe0-FAM dcProbe0FAM_G600_Bkgmult5 File:DcProbe0FAM 600 FAM ch00 PISA7Overlay Bkgmult5.png 364

Removing Background

  • dcProbe0-Cy3
 Ran PISA on NoDye_G680_Cy3_Bkgmult9 and dcProbe0Cy3_G550_Bkgmult3
 Saved bw images in workspace as backgroundCy3 and dcProbe0Cy3
 countOverlappedPoloniesv2(backgroundCy3,dcProbe0Cy3)
 ans = 0
 comp_img1=zeros(xsize,ysize,3);
 comp_img1(:,:,1)=dcProbe0Cy3;
 comp_img1(:,:,2)=backgroundCy3;
 imwrite(comp_img1,'dcProbe0Cy3vsbackgroundCy3.png');

File:DcProbe0Cy3vsbackgroundCy3.png

  • dcProbe0-FAM
 Ran PISA on NoDye_L2%_G680_FAM_Bkgmult9 and dcProbe0FAM_G600_Bkgmult5
 Saved bw images in workspace as backgroundFAM and dcProbe0FAM
 countOverlappedPoloniesv2(backgroundFAM,dcProbe0FAM)
 ans = 0
 comp_img1=zeros(xsize,ysize,3);
 comp_img1(:,:,1)=dcProbe0Cy3;
 comp_img1(:,:,2)=backgroundCy3;
 imwrite(comp_img1,'dcProbe0Cy3vsbackgroundCy3.png');

File:DcProbe0FAMvsbackgroundFAM.png

  • Conclusion: background is not a factor

Specificity of dcProbe0-Cy3 and dcProbe0-FAM

  • Is there overlap between the two? (There shouldn't be, they should be exclusive)
 countOverlappedPoloniesv2(dcProbe0Cy3,dcProbe0FAM)
 ans = 1

File:DcProbe0Cy3vsdcProbe0FAM.png

  • The only overlap is in the bottom right corner and looks more likely due to nearby rolonies than non-specific binding of both dyes to some structure
    • Red is dcProbe0-Cy3 and Green is dcProbe0-FAM

Overlay BrightField
File:DcProbe0Cy3vsdcProbe0FAM BF.png

Conclusion[edit]

Initial Imaging[edit]

  • Background signal did not play a factor since none overlapped with detected features during dcProbe0-Cy3 and dcProbe-FAM
    • We can say every feature we see is specific to 2nd rolony
  • dcProbe0-Cy3 and dcProbe0-FAM showed essentially no overlap, which gives us confidence these are true signals
    • Next time, we should be able to detect dcProbe1, dcProbe2, etc. and expect to see a fraction overlap with these
  • dcProbe0-Cy3 had 265 rolonies (~2.4/cell)
  • dcProbe0-FAM had 364 rolonies (~3.3/cell)
  • dcProbe0-Cy3(3641 probes) + dcProbe0-FAM (4097 probes) = 7,738 probes/12,964 total probes in Agi26k0gap = 60%
    • If these two dyes which can detect 60% of probes detected 629 2nd rolonies then we can approximate the total number of 2nd rolonies in this field of view = ~1050
  • There are ~9.5 2nd rolonies/cell