Matt:LabNotes/2015-9-29

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FISSEQ and DARTFISH[edit]

  • Prepare FISSEQ and DARTFISH samples from human brain tissue (BA8) for Harvard on glass slides
    • Two sections per glass slide
      • FISSEQ slide just do duplicates
      • DARTFISH slide do one without suppressorv2 (tissue close to frosted edge) and one with supperssorv2 (far from frosted edge)
  • Do one FISSEQ and one DARTFISH in culture dish as control
    • DARTFISH coverslip glass cracked

Protocol[edit]

Day 1[edit]

  1. Prepare 2 plastic culture dishes with hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH sterilize and UV
  2. Made 40ml 4% PFA at RT
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
  3. Take out BA8 section (2 glass slides and 2 coverslips) from -80C and incubate on 50C hot plate for 3min
  4. Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Submerge glass slides in 30ml 4%PFA in 50ml tube for 15min at 37C
  6. Wash with 2X SSPE by submerging
  7. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  8. Aspirate any liquid and attach gaskets to glass slides
  9. Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
  10. Wash with nuclease-free H2O three times
  11. Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
  12. Wash with nuclease-free PBS three times
    • Let sit in PBS for ~10min while preparing RT Mix
  13. Prepare 5X Reverse Transcription Mix on ice
Components Volume
H2O 157
10X M-MuLV Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
100uM N9 or 100uM FISSEQ_RT 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C
    • Put in pipette tip box with bottom filled with water and parafilmed around edges of box

Day 2[edit]

  1. Wash with 1X PBS once
    • FISSEQ Dish has very little solution left... but not bone dry
  2. Add 200ul BS(PEG)9 (20ul BS(PEG)9 + 980ul 1X PBS) and incubate 1hr at RT
    • Opened a new BS(PEG)9, couldn't see any solution in the vial but added 465ul DMSO just in case
    • THERE WAS NOTHING IN THE VIAL! AFTER 465ul DMSO ADDED FINAL VOLUME 465 WHEN SHOULD BE 565 AND NOT VISCOUS LIKE NORMAL
  3. Wash with 1X PBS twice
  4. Add Tris pH8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice
  6. RNA Removal
    • Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C
  7. Wash with H2O twice
  8. To DARTFISH slide: Add 100nM Padlock Probe Mix with 1000X suppv2 oligos in one sample and only 100nM Padlock Probe Mix in other and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr
    • Put in pipette tip box with bottom filled with water and parafilmed around edges of box
Component Volume
DEPC-H2O 33.43
Ampligase Buffer 10
Justin's Batch 4 7/13/2015 Padlock Probes 559nM]] 18
100nM suppv2 Oligos 28.57
Ampligase 10
Total 100
Component Volume
DEPC-H2O 62
Ampligase Buffer 10
Justin's Batch 4 7/13/2015 Padlock Probes 559nM]] 18
Ampligase 10
Total 100
  1. To FISSEQ slide and dish: Add 200ul CircLigaseII mix and incuabate 1hr at 60C
    • Put in pipette tip box with bottom filled with water and parafilmed around edges of box
Component Volume
DEPC-H2O 384
CircLigase Buffer 10X 60
MnCl2 50mM 30
Betaine 5M 120
CircLigase II (100U/ul) 6
Total 600
  1. Wash FISSEQ samples with PBS and store in PBS at 4C overnight

Day 3[edit]

  1. Wash with 1X PBS once
  2. To DARTFISH slide add 1uM FISSEQ_ppRCA (2ul 200uM FISSEQ_ppRCA + 398ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • DARTFISH slide was dried out
    • Edit: the slide may not have dried out so much as leaked. The slide has been leaking since this step.
      • Possible the 3M 200M adhesive is not resistant to long term high temp incubation.
  3. To FISSEQ slide and dish add 1uM FISSEQ_RCA (6ul 100uM FISSEQ_RCA + 594ul 2X SSC + 30% formamide) and incubate 1hr at 55C (should be 60C but only one oven)
    • Preheated to 70C before adding
  4. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  5. Add RCA mix and incubate 6hr at 30C
Component Volume
H2O 174
10X Phi29 Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
Phi 29 (low conc) 2
Total 200
  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 and store in 1X PBS at 4C

Imaging[edit]

  • FISSEQ rolonies in control dish
    • Density is low and spots are irregular shapes and sizes

File:20151002 BA8FISSEQforHarvard ControlDish Position1.jpg

File:20151002 BA8FISSEQforHarvard ControlDish Position2.jpg

Takeaways[edit]

  1. BS(PEG)9 vial was empty even though brand new, contact Fisher Sci for free replacement
  2. Attaching 22mmx22mm coverslip to bottom of 18mm diameter hole with 20mm diameter adhesive ring has too much room for error (leaking)
    • Cut 16mm holes in culture dishes and use 16mm diameter adhesive ring
    • Buy new adhesive (ARclad 7876) because when it gets old it becomes hard to peel off liner without messing up adhesive
      • Correction: Adhesives Research IS-8458-19 is the adhesive used
  3. 55-60C 20hr incubation dried out DARTFISH glass slide
    • Need foolproof method to prevent evaporation, maybe must use mineral oil layer on top
    • Edit: Leaking actually the problem in this case. Though 3M 300MP adhesive should be able to withstand 149C temperature for days