Editing
Matt:LabNotes/2014-9-6
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
=="Tertiary" Rolony Synthesis== *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=== *PGP1F sample from [[Hosuk:LabNotes/2014-4-14 | 4/14/14]] **[[Hosuk:LabNotes/2014-8-22 | 20X objective Full decoding]] and [[Hosuk:LabNotes/2014-8-29 | 63X objective Full decoding]] *Add 100ul Padlock Probe Mix (14pM per padlock probe) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 5 U/ul Ampligase||10 |- | 10X Ampligase Buffer||10 |- | [[Matt:LabNotes/2014-6-27 | 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/Β΅l) : 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
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Template used on this page:
Template:Table
(
edit
)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information