Revision as of 02:09, 21 October 2015 by >Mzcai
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]
- Prepare 2 plastic culture dishes with hole, tweezers, large plastic dish, and 50C hot plate
- Made 40ml 4% PFA at RT
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
- Take out BA8 section (2 glass slides and 2 coverslips) from -80C and incubate on 50C hot plate for 3min
- Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Submerge glass slides in 30ml 4%PFA in 50ml tube for 15min at 37C
- Wash with 2X SSPE by submerging
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Aspirate any liquid and attach gaskets to glass slides
- Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
- Wash with nuclease-free H2O three times
- Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
- Wash with nuclease-free PBS three times
- Let sit in PBS for ~10min while preparing RT Mix
- 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
|
- 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
- Wash with 1X PBS once
- FISSEQ Dish has very little solution left... but not bone dry
- 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
- Wash with 1X PBS twice
- Add Tris pH8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C
- Wash with H2O twice
- 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
|
- 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
|
- Wash FISSEQ samples with PBS and store in PBS at 4C overnight
- Wash with 1X PBS once
- 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.
- 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
- Wash with 2X SSC once, 1X SSC once, and 1X PBS once
- 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
|
- Wash with 1X PBS once
- Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate at RT for 1hr
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate at RT for 30min
- 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]
- BS(PEG)9 vial was empty even though brand new, contact Fisher Sci for free replacement
- 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
- 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