Matt:LabNotes/2016-7-19

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Revision as of 19:44, 23 July 2016 by >Mzcai (→‎Day 2)
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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

  1. Fix embryo in 4% PFA at 37C for 15min
  2. 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)
  3. Wash with 1X PBS
  4. Incubate embryo in solution at 4C overnight (20hrs)
  5. Polymerize gel by incubating at 37C for 3hr
    • Did not degas under Argon gas but can do next time if not polymerized enough
  6. 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
  7. Remove embryo from gel
    • Pipette away surrounding hydrogel and wash with 1X PBS
  8. Incubate in 8% SDS at 37C on rotator for overnight
    • Tissue will not be fully clear until put into refractive index matching media
  9. 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

  1. 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
  2. Incubate embryo in solution at 4C overnight (20hrs)
  3. Polymerize gel by incubating at 37C for 3hr
  4. 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
  5. Remove embryo from gel
    • Pipette away surrounding hydrogel and wash with 1X PBS
    • Roll on kimwipe to remove bits of gel
  6. Incubate in 8% SDS at 37C on rotator for overnight
    • Tissue will not be fully clear until put into refractive index matching media
  7. 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

  1. Wash with PBS three times
  2. 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
  1. Add 100ul to each and incubate 10min at 4C and then ~16hr at 37C

Day 2

  1. Wash with 1X PBS once
  2. Add 100ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  5. Wash with 1X PBS twice
  6. Add RNase H mix and incubate 1hr at 37C
    • H2O - 168ul
    • RNase H Buffer - 20ul
    • Riboshredder - 2ul
    • RNase H - 10ul
  7. Wash with nuclease-free H2O twice
  8. 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
  9. Wash with 1X PBS twice
    • Lost many embryos due to sticking to pipette tip and unable to get off without scraping
    • Only one CLARITY and one PACT embryo left
  10. Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
  11. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
    • PACT embryo stuck to pipette tip so cut off the tip and put in tube for incubation
  12. 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
  13. Add RCA mix and incubate at 30C overnight (~12hrs)

Day 3

  1. Wash with 1X PBS once
    • Could not find embryo stuck to pipette tip
    • Only one CLARITY embryo left
  2. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  5. Wash with 1X PBS twice