Matt:LabNotes/2014-9-6: Difference between revisions
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===Conclusion=== | ===Conclusion=== | ||
*No improvement over | *No improvement over [[Hosuk:LabNotes/2014-8-29 | 63X objective Full decoding of 2nd rolonies]] | ||
**Didn't look at identical position but since results were slightly worse for both positions here, suggests this method is not effective | **Didn't look at identical position but since results were slightly worse for both positions here, suggests this method is not effective |
Latest revision as of 02:44, 28 September 2014
"Tertiary" Rolony Synthesis[edit]
- Hypothesis: We can increase the number of gene-specific rolonies (previously known as secondary rolonies) by repeating padlock probe capture and rolonization on samples with primary and secondary rolonies
- There should be more targets for the padlock probes to capture since all primary rolonies are still fixed and 100% of secondary rolonies will be possible targets for padlock probes
- May increase amplification bias
Procedure[edit]
- PGP1F sample from 4/14/14
- Add 100ul Padlock Probe Mix (14pM per padlock probe)
Components | Volume |
5 U/ul Ampligase | 10 |
10X Ampligase Buffer | 10 |
908 nM Agi26k0gap | 18.6 |
H2O | 61.4 |
Total | 100 |
- Incubate at 37C for 30min
- Incubate at 45C for 26hrs
- Wash with 1X PBS twice
- Add 200ul 0.5 uM FISSEQ_ppRCA primer in 2X SSC (pre-heated to 75C) and incubate at 45C for 30min
- Wash with 2X SSC twice
- Add 200ul RCA Mix and incubate at 30C for 16hrs
- H2O : 172uL
- 10x Buffer : 20uL
- dNTP : 2uL
- a-dUTP : 4uL
- Phi29 (100 U/µl) : 2uL
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
- Wash with 1X PBS twice
- Add 200ul 1M Tris pH 8.0 at RT for 30min
- Wash with 1X PBS twice and store in PBS
7 Cycle 3-Dye Decoding[edit]
- Saved in 9-9-2014
- Used 0.5uM of each dcProbe
- For each cycle, 3 dcProbes were added together
Image Analysis[edit]
- Hosuk aligned and cropped images
- One fluorescent image is selected from each cycle to use for aligning and stored in directory: '0.Align'
- All the aligned images stored in directory: '1.Shift'
- All the aligned and cropped images stored in directory: '1.MIP'
- Made MIP of aligned and cropped images
- Filename prefix: MIP_Crop_MIP_
- Stored in directory: '2.AllMIP'
- Run PISA on MIP_Crop_MIP_
- Chose settings to maximize number of features
- Gaussian std: 2
- upper bound: -2e-4
- area upper: 200
- area lower: 8
- axratio lower: .6
- circ upper: 1.6
- circ lower: .8
- perim conn: 8
- bkgmult lower: 3
- Save BW file with prefix: BW_MIP_Crop_MIP_
- Chose settings to maximize number of features
- Run MasterRun.m in Matlab_FullDecode_PISAMask/FISSEQ_Decoding
- Put together by Hosuk
- immultiply BW mask with each aligned and cropped image and stores output in '3.Masked'
- For every 'feature location' in each of the 21 images, calculates average intensity and compares to the other dyes
- Use a normalized threshold of 0.2 to determine 'No Signal' during that cycle
Results[edit]
Position 1[edit]
- 3,325 total features counted in PISA (237.5/cell)
- 221 features had barcodes that mapped to rolonies (15.8/cell)
- Mapped barcodes represent 37 different genes
Gene | Barcode | Num |
COL1A1 | 3013130 | 113 |
GREM1 | 332120 | 34 |
GEMIN2 | 3020131 | 17 |
HAT1 | 3020212 | 9 |
IGSF10 | 1013130 | 7 |
RTN4 | 2033012 | 4 |
VAX2 | 2320102 | 3 |
RUNX1 | 3021203 | 2 |
GSK3B | 3020211 | 2 |
TMEM176A | 2232200 | 2 |
HNF1A | 3020231 | 2 |
SOHLH2 | 2130203 | 1 |
CLCN7 | 132110 | 1 |
AHNAK | 21312 | 1 |
RPLP0P2 | 2032120 | 1 |
SERPINA12 | 2101202 | 1 |
NUMB | 3021101 | 1 |
PLEK2 | 3021130 | 1 |
IL1RN | 1020131 | 1 |
HOXD1 | 1010131 | 1 |
RUNX2 | 3021210 | 1 |
ABHD12B | 11233 | 1 |
EPHB4 | 232012 | 1 |
SLC17A5 | 2110201 | 1 |
ERBB3 | 3013330 | 1 |
PLEKHA4 | 2010211 | 1 |
ITGB4 | 3020331 | 1 |
FOXA1 | 3020112 | 1 |
HTR1B | 1011130 | 1 |
CDH1 | 3013110 | 1 |
PSG11 | 2020131 | 1 |
KPRP | 1033130 | 1 |
FAM196A | 301222 | 1 |
FAM38A | 301322 | 1 |
ZNF649 | 3001222 | 1 |
CCDC88C | 120222 | 1 |
CDR1 | 130113 | 1 |
- PISA segmentation to identify features (red) from raw image (blue)
File:MIP Crop MIP PGP1F S2 2ndRol20140414 Decode3rd PISA7Overlay 3325.png
- Tetraplot of average intensities does not look good
File:20140909 Tetraplot Pos1.PNG
- Red: Cy5
- Green: Cy3
- Blue: Alexa-488
Position 2[edit]
- 3,610 total features counted in PISA (278/cell)
- 342 features had barcodes that mapped to rolonies (26.3/cell)
- Mapped barcodes represent 49 different genes
Gene | Barcode | Num |
COL1A1 | 3013130 | 172 |
GEMIN2 | 3020131 | 29 |
HAT1 | 3020212 | 23 |
RUNX1 | 3021203 | 13 |
COL1A2 | 3013201 | 12 |
IL22 | 1020212 | 6 |
NUMB | 3021101 | 5 |
ANXA5 | 31122 | 5 |
ERBB3 | 3013330 | 4 |
CLDN20 | 132210 | 3 |
PROX1 | 2013120 | 3 |
GRB7 | 3020133 | 3 |
CDH1 | 3013110 | 3 |
GSK3B | 3020211 | 3 |
GREM1 | 332120 | 3 |
CTNNA1 | 3013210 | 2 |
APOB48R | 31222 | 2 |
IL1RN | 1020131 | 2 |
PRSS35 | 2013320 | 2 |
CHAT | 3013120 | 2 |
RUNX2 | 3021210 | 2 |
TAT | 2211100 | 2 |
POU1F1 | 2011130 | 1 |
KAT2B | 3020332 | 1 |
PRSS38 | 2013330 | 1 |
ITGB4 | 3020331 | 1 |
GAP43 | 320332 | 1 |
PLEK2 | 3021130 | 1 |
AHNAK | 21312 | 1 |
IGSF10 | 1013130 | 1 |
RASAL3 | 2022320 | 1 |
PRG2 | 2012320 | 1 |
CCDC62 | 120122 | 1 |
DDX4 | 3013230 | 1 |
IL1B | 1020121 | 1 |
IL29 | 1020233 | 1 |
GFAP | 3020132 | 1 |
IGF2BP2 | 3020233 | 1 |
STX1B | 2202102 | 1 |
RPRM | 2032130 | 1 |
FGFR1 | 3020111 | 1 |
PROX2 | 2013130 | 1 |
CCDC144NL | 113202 | 1 |
POU4F3 | 2011230 | 1 |
KCNE2 | 1023130 | 1 |
EGF | 3013310 | 1 |
TGFB1 | 2221300 | 1 |
LGALS1 | 1103023 | 1 |
OMD | 1231100 | 1 |
- PISA segmentation to identify features (red) from raw image (blue)
File:MIP Crop MIP PGP1F S2 2ndRol20140414 Decode3rd PISA7Overlay 3610.png
- Tetraplot of average intensities does not look good
File:20140909 Tetraplot Pos2.PNG
- Red: Cy5
- Green: Cy3
- Blue: Alexa-488
Conclusion[edit]
- No improvement over 63X objective Full decoding of 2nd rolonies
- Didn't look at identical position but since results were slightly worse for both positions here, suggests this method is not effective