Matt:LabNotes/2016-3-14

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DARTFISH on BA8 Sections with PA gel[edit]

Protocol[edit]

Day 1[edit]

  1. Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Used 4% PFA from 3/8/2016
  3. Take out second best remaining BA8 sections (4th from back) from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  8. Wash with cold nf-H2O three times and check for degradation
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with nf-1X PBS three times and check for degradation
  11. Add 75ul gel casting mix filtered and degassed
    • 4% PFA, 10% Acrylamide/Bis-Acrylamide (199:1), 0.2% BSA, 0.1% APS, 0.1% TEMED
  12. Cover with plastic coverslips cut and glued together such that it leaves ~0.25mm space for gel height
  13. Seal in plastic bag and vacuum out air before filling with argon
  14. Let sit at RT for 30min
  15. Aspirate non-polymerized gel and wash once with cold 1X SSPE
    • Edges did not polymerize but the grey matter is fully covered
  16. 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 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C

Day 2[edit]

  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice
  6. RNA Removal
    • Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  7. Wash with nf-H2O twice
  8. Prepare Ampligase mix on ice
    • Preheat H2O + Padlock probes + suppv2 Oligos to 85C and then snap cool before adding Ampligase buffer and enzyme
  • B9: 216nM
Component Volume
DEPC-H2O 5.1
Ampligase Buffer 10
Justin's Batch 9 9/11/2015 Padlock Probes 216nM]] 46.3
100nM suppv2 Oligos 28.57
Ampligase 10
Total 100
  1. Add mix to sample and incubate for 30min at 37C
  2. Move sample to 60C and oven slowly decreases to 55C and held for ~24hr

Day 3[edit]

  • Center of gel was not covered with liquid when taking out
  1. Wash with 1X PBS once
  2. Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 70C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. Add RCA mix and incubate 15hr at 30C
Component Volume
H2O 174
10X Phi29 Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
Phi 29 (low conc) 2
Total 200

Day 4[edit]

  1. Wash with 1X PBS once
  2. Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate at RT for 1hr
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate at RT for 30min
  5. Wash with 1X PBS twice

Check Rolony[edit]

  1. Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C
  2. Incubate for 10min at RT
  3. Wash with 2X SSC twice
  4. Image (saved in 3-18-2016)
    • As usual have the tissue oriented like a backwards 'P'
    • Image on horizontal line at widest section of 'P'
      • Edge is right side of backwards 'P' (ie the vertical edge)
      • Take images every certain distance from edge
    • Bottom of P wasn't covered by gel so image that as well

Results[edit]

  • Images not shown because no rolonies anywhere
  • Only some autofluorescence or non-specific fluorescence

Conclusion[edit]

  • My guess is that the 4% PFA in hydrogel solution over cross-linked RNA during gel polymerization?
  • This prevents reverse transcription