Matt:LabNotes/2014-9-6: Difference between revisions
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**[[Hosuk:LabNotes/2014-8-22 | 20X objective Full decoding]] and [[Hosuk:LabNotes/2014-8-29 | 63X objective Full decoding]] | **[[Hosuk:LabNotes/2014-8-22 | 20X objective Full decoding]] and [[Hosuk:LabNotes/2014-8-29 | 63X objective Full decoding]] | ||
*Add 100ul Padlock Probe Mix | *Add 100ul Padlock Probe Mix (14pM per padlock probe) | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
Line 28: | Line 28: | ||
*Add 200ul 0.5 uM FISSEQ_ppRCA primer in 2X SSC (pre-heated to 75C) and incubate at 45C for 30min | *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 | *Wash with 2X SSC twice | ||
*Add 200ul RCA Mix and incubate at 30C for | *Add 200ul RCA Mix and incubate at 30C for 16hrs | ||
**H2O : 172uL | **H2O : 172uL | ||
**10x Buffer : 20uL | **10x Buffer : 20uL | ||
Line 34: | Line 34: | ||
**a-dUTP : 4uL | **a-dUTP : 4uL | ||
**Phi29 (100 U/µl) : 2uL | **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=== | |||
*Saved in 9-9-2014 | |||
*Used 0.5uM of each dcProbe | |||
**For each cycle, 3 dcProbes were added together | |||
===Image Analysis=== | |||
*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_ | |||
*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=== | |||
====Position 1==== | |||
*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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Gene''' | |||
| align="center" style="background:#f0f0f0;"|'''Barcode''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | 650px]] | |||
*Tetraplot of average intensities does not look good | |||
[[File:20140909_Tetraplot_Pos1.PNG | 650px]] | |||
*Red: Cy5 | |||
*Green: Cy3 | |||
*Blue: Alexa-488 | |||
====Position 2==== | |||
*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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Gene''' | |||
| align="center" style="background:#f0f0f0;"|'''Barcode''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | 650px]] | |||
*Tetraplot of average intensities does not look good | |||
[[File:20140909_Tetraplot_Pos2.PNG | 650px]] | |||
*Red: Cy5 | |||
*Green: Cy3 | |||
*Blue: Alexa-488 | |||
===Conclusion=== | |||
*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 |
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