Matt:LabNotes/2016-8-3
Jump to navigation
Jump to search
DARTFISH on BA8 with and without PA Gel
- 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
Day 1
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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