Matt:LabNotes/2016-7-19
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CLARITY and PACT for FISSEQ in Mouse Embryo E7.5
- Need more permeabilization to get enzymes and reagents deeper into tissue
- To permeabilize will remove lipids with detergent in long incubation
- Need to embed tissue in hydrogel matrix to create structure that will survive detergent
Experiment
PACT Protocol
- Fix embryo in 4% PFA at 37C for 15min
- Make 4mL PACT solution on ice
- 4% acrylamide in 1X PBS plus 0.25% VA-044 (degassed)
- 3.584mL MilliQ H2O + 0.448mL 10X PBS + 0.448mL 40% acrylamide (cold) + 0.0112g 0.25% VA-044 (cold)
- Wash with 1X PBS
- Incubate embryo in solution at 4C overnight (20hrs)
- Polymerize gel by incubating at 37C for 3hr
- Did not degas under Argon gas but can do next time if not polymerized enough
- Make 500mL 8% SDS solution
- Could use 0.2 M boric acid or 0.01 M PBS (1X)
- 6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS
- 50mL = 5mL 10X PBS + 20mL 20% SDS + 25mL H2O
- Remove embryo from gel
- Pipette away surrounding hydrogel and wash with 1X PBS
- Incubate in 8% SDS at 37C on rotator for overnight
- Tissue will not be fully clear until put into refractive index matching media
- Wash embryo in PBS + 0.1% TX-100 at 37C on a rotator for 6 hrs
- Need to wash away SDS or it will precipitate forming cloudy spots
CLARITY Protocol
- Make 4mL CLARITY solution on ice
- 4% PFA, 0.95% acrylamide, 0.05% bis-acrylamide, 0.25% VA-044, 1X PBS (degassed)
- 0.5mL 40% PFA + 0.125mL 40% 19:1 acrylamide:bis + 0.0125g VA-044 + 0.5mL 10X PBS + 3.875mL H2O
- Incubate embryo in solution at 4C overnight (20hrs)
- Polymerize gel by incubating at 37C for 3hr
- Make 500mL 8% SDS solution
- Could use 0.2 M boric acid or 0.01 M PBS (1X)
- 6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS
- 50mL = 5mL 10X PBS + 20mL 20% SDS + 25mL H2O
- Remove embryo from gel
- Pipette away surrounding hydrogel and wash with 1X PBS
- Roll on kimwipe to remove bits of gel
- Incubate in 8% SDS at 37C on rotator for overnight
- Tissue will not be fully clear until put into refractive index matching media
- Wash embryo in PBS + 0.1% TX-100 at 37C on a rotator for 6 hrs
- Need to wash away SDS or it will precipitate forming cloudy spots
FISSEQ
- 4 tdTomato positive embryos were taken by Paola for confocal/two-photon microscopy test
- For tdTomato negative embryos do FISSEQ
- 4 CLARITY embryos and 2 PACT
Day 1
- Wash with PBS three times
- Prepare 3X Reverse Transcription Mix on ice
Components | Volume |
H2O | 157 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM FISSEQ_RT | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Add 100ul to each and incubate 10min at 4C and then ~16hr at 37C
Day 2
- Wash with 1X PBS once
- Add 100ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 200ul 1M Tris (pH 8.0) for 30min at RT
- Wash with 1X PBS twice
- Add RNase H mix and incubate 1hr at 37C
- H2O - 168ul
- RNase H Buffer - 20ul
- Riboshredder - 2ul
- RNase H - 10ul
- Wash with nuclease-free H2O twice
- Add CircLigase mix and incubate 3hr at 60C
- H2O - 128ul
- CircLigase Buffer 10X - 20ul
- MnCl2 50mM - 10ul
- Betaine 5M - 40ul
- CircLigase II 100U/ul - 2ul
- Wash with 1X PBS twice
- Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
- Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
- Prepare RCA reaction mix on ice
- H2O - 174ul
- Phi29 Buffer 10X - 20ul
- dNTP 25mM - 2ul
- aa-dUTP 4mM - 2ul
- Phi29 DNA polymerase 100U/ul - 2ul
- Add RCA mix and incubate at 30C overnight (~15hrs)