Matt:LabNotes/2016-8-3: Difference between revisions
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| 4mM aa-dUTP||2 | | 4mM aa-dUTP||2 | ||
|- | |- | ||
| 100uM | | 100uM N9 primer||5 | ||
|- | |- | ||
| RNase Inhibitor||2 | | RNase Inhibitor||2 | ||
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#Add mix to sample and incubate for 30min at 37C | #Add mix to sample and incubate for 30min at 37C | ||
#Move sample to 60C and oven slowly decreases to 55C and held for ~ | #Move sample to 60C and oven slowly decreases to 55C and held for ~44hr | ||
===Day 3=== | ===Day 3=== | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
#Add 200ul (1ul 200uM | #Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C | ||
#Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS | #Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS | ||
#Prepare RCA reaction mix on ice | #Prepare RCA reaction mix on ice | ||
Line 182: | Line 181: | ||
#Incubate for 10min at RT | #Incubate for 10min at RT | ||
#Wash with 2X SSC twice | #Wash with 2X SSC twice | ||
#Image (saved in | #Image (saved in 8-8-2016) | ||
====Notes==== | |||
*Laser intensity = 1%, Cy3 gain = 600 (tried increasing gain but only increased background) | |||
*Z-stack system optimized for 63X objective (0.30um step size) | |||
*Imaged Samples 1, 2, and 3 in that order | |||
*Imaged each from bottom of white matter to top-left of white matter | |||
*Sample 1 also imaged up-left of white matter to show degradation and lack of rolonies in grey matter | |||
*Sample 1 had thickness <10um, thicker in white matter obviously | |||
*Sample 2 shows odd round objects in BF image when in white matter area | |||
**For some reason these are not seen in areas that are definite grey matter but I am not sure where the white/grey matter boundary is | |||
*Sample 3 had thickest z-stack probably because gel was made thicker and rolonies were generated in gel but above tissue | |||
====Bottom==== | |||
====Bottom-Mid==== | |||
====Mid==== | |||
====Top Left==== | |||
====Grey Matter==== | |||
===Decoding Sample 2: Imaging=== | |||
====Add [[Matt:LabNotes/2015-8-13#Add_Beads| 1:2000 diluted Fiducial Beads]]==== | |||
#Strip with pre-heated 80% formamide twice for 15 min each | |||
#Wash with 1X PBS twice | |||
#Wash with 10mM HEPES | |||
#Get aliquoted 1:2000 diluted beads from dcProbe box in 4C | |||
#Vortex (normally should sonicate) | |||
#Add to sample and incubate 5min at RT | |||
#Wash with 10mM HEPES once | |||
#Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 20mM HEPES) and incubate at RT for 1hr | |||
#Wash with 1X PBS twice | |||
#Add 1M Tris pH 8.0 and incubate at RT for 30min | |||
*Did not detect any beads, may need to allow longer incubation due to gel | |||
====Notes==== | |||
*22 images 170um away from each other | |||
**184umx184um FOV so ~14um (<10%) overlap between images | |||
*By the time I finish defining z-start and z-end positions the stage has drifted | |||
**Seems like stage drifts down ~3um per hour | |||
**Need to define >3um below what I want and leave extra room above and below | |||
*Left sample overnight and it dried out (gel was all cracked) | |||
**Rehydrated in 2X SSC for 1hr | |||
***Tissue morphology looks the same | |||
***Fewer/less rolony signal but this could be due to 2 other factors | |||
*20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle# | |||
**Cycle0 = Universal rolony probe (Cy3) | |||
**Cycle1-6 for decoding | |||
**Cycle7 for DRAQ5 | |||
*Pos1 of 22 with approx position of tissue section outlined in red | |||
[[File:20160810_22pos_DARTFISH_BA8_Pos1.JPG.JPG|350px]] | |||
*Pos22 of 22 with approx position of imaged tile outlined in red | |||
[[File:20160810_22pos_DARTFISH_BA8_Pos22.JPG|350px]] | |||
=====Cycle2===== | |||
*Retook Position19019 and Position20020 because z range was offset, started from middle of signal | |||
=====Cycle6===== | |||
*Retook Position20020 and Position21021 because z range was offset, started from middle of signal | |||
===Decoding Sample 2: Image Analysis=== | |||
*331 GB total size of raw images | |||
*Due to time constraints during imaging, could not export raw tiff files from Leica software during imaging process | |||
**Each cycle would need >30min to export and I already use all the downtime to save the experiment in Leica format | |||
#So first thing to do was reopen each experiment in Leica and then export as tiff | |||
#*Each cycle saved as its own experiment because Leica software has limited memory | |||
#*Each cycle takes about 2 hours to open and then export | |||
#Then uploaded raw data from local computer to ConfocalStore server | |||
#Then downloaded from ConfocalStore server to my laptop's external hard drive | |||
#Replaced Cycle2 Pos19 & Pos20 and Cycle6 Pos20 & Pos21 with retaken images | |||
#*i.e. replace 20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle2\Position19019\ with 20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle2_Pos19-20\Position19019\ | |||
#First try 2D decoding because much faster | |||
#Make Max Intensity Z Projection for every stack | |||
#*[[Media:BatchMIP.txt|BatchMIP.py]] | |||
<!-- | |||
==Results== | ==Results== | ||
--> | --> |
Latest revision as of 19:49, 20 August 2016
DARTFISH on BA8 with and without PA Gel[edit]
- Try 3 samples with polyacrylamide gel added at different points in protocol
- Sample 1: No gel
- Sample 2: Gel added after permeabilization
- Sample 3: Gel added after RNA removal
- Used 200nM padlock probe (twice the normal concentration)
- Gel mix and protocol is same as this with BS(PEG)9
Component | Volume |
40% AB 199:1 Mix | 6.25 |
10% BSA | 1 |
1mM Acryd/Amine Linker | 6.25 |
H2O | 33.5 |
5% TEMED | 1 |
5% APS | 1 |
BS(PEG)9 | 1 |
Total | 50 |
Protocol[edit]
Day 1[edit]
- Prepare 3 plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Make 4% PFA in 1X PBS
- 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
- Take out BA8 section from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times and check for degradation
- Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with nf-1X PBS three times and check for degradation
To Sample 2 Only[edit]
- Skip these steps and go to Reverse Transcription Immediately for other 2 samples
- Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
- Secure in plastic jig
- Add 50ul gel casting mix filtered and degassed
- 5% gel with 125uM Acrydite-Amine linker and 5mM BS(PEG)9
- Seal in plastic bag and vacuum out air before filling with argon
- Let sit at RT for 30min
- Aspirate non-polymerized gel and wash once with 1X PBS
- Attach coverslip to bottom of petri dish
- Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X PBS
Continue for All Samples[edit]
- Attach coverslip to bottom of petri dish
- Prepare Reverse Transcription Mix on ice and add
Components | Volume |
H2O | 159 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM N9 primer | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~15hr at 37C
Day 2[edit]
- Wash with 1X PBS once
- Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
- Wash with nf-H2O twice
To Sample 3 Only[edit]
- Add 50ul gel casting mix filtered and degassed
- 5% gel with 125uM Acrydite-Amine linker, 5mM BS(PEG)9, and Use 478nM TB12kApr2016 V4 Probes instead of H2O (33.5ul)
- Seal in plastic bag and vacuum out air before filling with argon
- Let sit at RT for 30min
- Aspirate non-polymerized gel and wash once with 1M Tris
- Prepare Ampligase mix on ice
- Preheat Padlock Probes then snap cool before adding Ampligase buffer and enzyme
Component | Volume |
Ampligase Buffer | 10 |
478nM | 80 |
Ampligase | 10 |
Total | 100 |
- Add mix to sample and incubate for 30min at 37C
- Move sample to 60C and oven slowly decreases to 55C and held for ~24hr
To Samples 1 & 2[edit]
- Prepare Ampligase mix on ice
- Preheat Padlock Probes then snap cool before adding Ampligase buffer and enzyme
Component | Volume |
Ampligase Buffer | 10 |
478nM | 80 |
Ampligase | 10 |
Total | 100 |
- Add mix to sample and incubate for 30min at 37C
- Move sample to 60C and oven slowly decreases to 55C and held for ~44hr
Day 3[edit]
- Wash with 1X PBS twice
- Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C
- Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
- Prepare RCA reaction mix on ice
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
- Add RCA mix and incubate at 30C overnight (~15hrs)
Day 4[edit]
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate at RT for 30min
- Wash with 1X PBS twice
Imaging[edit]
- Add 0.5uM FISSEQ_Adpt in 30% formamide + 2X SSC preheated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice
- Image (saved in 8-8-2016)
Notes[edit]
- Laser intensity = 1%, Cy3 gain = 600 (tried increasing gain but only increased background)
- Z-stack system optimized for 63X objective (0.30um step size)
- Imaged Samples 1, 2, and 3 in that order
- Imaged each from bottom of white matter to top-left of white matter
- Sample 1 also imaged up-left of white matter to show degradation and lack of rolonies in grey matter
- Sample 1 had thickness <10um, thicker in white matter obviously
- Sample 2 shows odd round objects in BF image when in white matter area
- For some reason these are not seen in areas that are definite grey matter but I am not sure where the white/grey matter boundary is
- Sample 3 had thickest z-stack probably because gel was made thicker and rolonies were generated in gel but above tissue
Bottom[edit]
Bottom-Mid[edit]
Mid[edit]
Top Left[edit]
Grey Matter[edit]
Decoding Sample 2: Imaging[edit]
Add 1:2000 diluted Fiducial Beads[edit]
- Strip with pre-heated 80% formamide twice for 15 min each
- Wash with 1X PBS twice
- Wash with 10mM HEPES
- Get aliquoted 1:2000 diluted beads from dcProbe box in 4C
- Vortex (normally should sonicate)
- Add to sample and incubate 5min at RT
- Wash with 10mM HEPES once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 20mM HEPES) and incubate at RT for 1hr
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate at RT for 30min
- Did not detect any beads, may need to allow longer incubation due to gel
Notes[edit]
- 22 images 170um away from each other
- 184umx184um FOV so ~14um (<10%) overlap between images
- By the time I finish defining z-start and z-end positions the stage has drifted
- Seems like stage drifts down ~3um per hour
- Need to define >3um below what I want and leave extra room above and below
- Left sample overnight and it dried out (gel was all cracked)
- Rehydrated in 2X SSC for 1hr
- Tissue morphology looks the same
- Fewer/less rolony signal but this could be due to 2 other factors
- Rehydrated in 2X SSC for 1hr
- 20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle#
- Cycle0 = Universal rolony probe (Cy3)
- Cycle1-6 for decoding
- Cycle7 for DRAQ5
- Pos1 of 22 with approx position of tissue section outlined in red
File:20160810 22pos DARTFISH BA8 Pos1.JPG.JPG
- Pos22 of 22 with approx position of imaged tile outlined in red
File:20160810 22pos DARTFISH BA8 Pos22.JPG
Cycle2[edit]
- Retook Position19019 and Position20020 because z range was offset, started from middle of signal
Cycle6[edit]
- Retook Position20020 and Position21021 because z range was offset, started from middle of signal
Decoding Sample 2: Image Analysis[edit]
- 331 GB total size of raw images
- Due to time constraints during imaging, could not export raw tiff files from Leica software during imaging process
- Each cycle would need >30min to export and I already use all the downtime to save the experiment in Leica format
- So first thing to do was reopen each experiment in Leica and then export as tiff
- Each cycle saved as its own experiment because Leica software has limited memory
- Each cycle takes about 2 hours to open and then export
- Then uploaded raw data from local computer to ConfocalStore server
- Then downloaded from ConfocalStore server to my laptop's external hard drive
- Replaced Cycle2 Pos19 & Pos20 and Cycle6 Pos20 & Pos21 with retaken images
- i.e. replace 20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle2\Position19019\ with 20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle2_Pos19-20\Position19019\
- First try 2D decoding because much faster
- Make Max Intensity Z Projection for every stack