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

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*Pos1 of 22 with approx position of tissue section outlined in red
*Pos1 of 22 with approx position of tissue section outlined in red
[[File:20160810_22pos_DARTFISH_BA8_Pos1.JPG.JPG|350px]]
[[File:20160810_22pos_DARTFISH_BA8_Pos1.JPG.JPG|350px]]
*Pos22 of 22
*Pos22 of 22 with approx position of imaged tile outlined in red
[[File:20160810_22pos_DARTFISH_BA8_Pos22.JPG|350px]]
[[File:20160810_22pos_DARTFISH_BA8_Pos22.JPG|350px]]
=====Cycle2=====
=====Cycle2=====

Revision as of 19:32, 18 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: Imaging

Add 1:2000 diluted Fiducial Beads

  1. Strip with pre-heated 80% formamide twice for 15 min each
  2. Wash with 1X PBS twice
  3. Wash with 10mM HEPES
  4. Get aliquoted 1:2000 diluted beads from dcProbe box in 4C
  5. Vortex (normally should sonicate)
  6. Add to sample and incubate 5min at RT
  7. Wash with 10mM HEPES once
  8. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 20mM HEPES) and incubate at RT for 1hr
  9. Wash with 1X PBS twice
  10. Add 1M Tris pH 8.0 and incubate at RT for 30min
  • Did not detect any beads, may need to allow longer incubation due to gel

Notes

  • 22 images 170um away from each other
    • 184umx184um FOV so ~14um (<10%) overlap between images
  • By the time I finish defining z-start and z-end positions the stage has drifted
    • Seems like stage drifts down ~3um per hour
    • Need to define >3um below what I want and leave extra room above and below
  • Left sample overnight and it dried out (gel was all cracked)
    • Rehydrated in 2X SSC for 1hr
      • Tissue morphology looks the same
      • Fewer/less rolony signal but this could be due to 2 other factors
  • 20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle#
    • Cycle0 = Universal rolony probe (Cy3)
    • Cycle1-6 for decoding
    • Cycle7 for DRAQ5
  • Pos1 of 22 with approx position of tissue section outlined in red

File:20160810 22pos DARTFISH BA8 Pos1.JPG.JPG

  • Pos22 of 22 with approx position of imaged tile outlined in red

File:20160810 22pos DARTFISH BA8 Pos22.JPG

Cycle2
  • Retook Position19019 and Position20020 because z range was offset, started from middle of signal
Cycle6
  • Retook Position20020 and Position21021 because z range was offset, started from middle of signal

Decoding Sample 2: Image Analysis

  • 331 GB total size of raw images
  • Due to time constraints during imaging, could not export raw tiff files from Leica software during imaging process
    • Each cycle would need >30min to export and I already use all the downtime to save the experiment in Leica format
  1. So first thing to do was reopen each experiment in Leica and then export as tiff
    • Each cycle saved as its own experiment because Leica software has limited memory
    • Each cycle takes about 2 hours to open and then export
  2. Then uploaded raw data from local computer to ConfocalStore server
  3. Then downloaded from ConfocalStore server to my laptop's external hard drive
  4. Replaced Cycle2 Pos19 & Pos20 and Cycle6 Pos20 & Pos21 with retaken images
    • i.e. replace 20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle2\Position19019\ with 20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle2_Pos19-20\Position19019\
  5. First try 2D decoding because much faster
  6. Make Max Intensity Z Projection for every stack