Matt:LabNotes/2016-8-3: Difference between revisions

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*Sample 2 shows odd round objects in BF image when in white matter area
*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
**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


===Decoding Sample 2===
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==Results==
==Results==


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Revision as of 22:47, 9 August 2016

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)
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

  1. Prepare 3 plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Make 4% PFA in 1X PBS
    • 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
  3. Take out BA8 section from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  7. Wash with cold nf-H2O three times and check for degradation
  8. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  9. 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
  1. Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
    • Secure in plastic jig
  2. Add 50ul gel casting mix filtered and degassed
    • 5% gel with 125uM Acrydite-Amine linker and 5mM BS(PEG)9
  3. Seal in plastic bag and vacuum out air before filling with argon
  4. Let sit at RT for 30min
  5. Aspirate non-polymerized gel and wash once with 1X PBS
  6. Attach coverslip to bottom of petri dish
  7. Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X PBS

Continue for All Samples

  1. Attach coverslip to bottom of petri dish
  2. 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
  1. Incubate 10min at 4C and then ~15hr at 37C

Day 2

  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

To Sample 3 Only

  1. Add 50ul gel casting mix filtered and degassed
  2. Seal in plastic bag and vacuum out air before filling with argon
  3. Let sit at RT for 30min
  4. Aspirate non-polymerized gel and wash once with 1M Tris
  5. 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
  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

To Samples 1 & 2

  1. 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
  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 ~44hr

Day 3

  1. Wash with 1X PBS twice
  2. Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C
  3. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  4. 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
  1. Add RCA mix and incubate at 30C overnight (~15hrs)

Day 4

  1. Wash with 1X PBS once
  2. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 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

Imaging

  1. Add 0.5uM FISSEQ_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 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

Decoding Sample 2