Matt:LabNotes/2016-8-18
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Mouse Brain FISSEQ test
- Repeat yesterday's experiment but with way less pepsin
Previous mouse brain FISSEQ tries
2nd try
3rd try
4th try
7th try
9th try
4X BF Image of mouse brain sections from Yun
Sample Info
- Received 10 x 10um thick sections of mouse brain on Vectabond and Bind-Silane treated coverglass
- Each section is half of coronal section
- Fresh frozen tissue, cryostat into 10um sections on vectabond coverslips, dry for 30min at -20C in cryostat chamber
- Box up and freeze at -80C
- Coverslip attached to glass slide by a drop of frozen water
FISSEQ Protocol
Summary
- Try without hydrogel, hopefully no degradation
- Try 3 samples with 3 different pepsin incubation times
- 1 min
- 5 min
- 10 min
Day 1
- Prepare 3 plastic culture dishes with 10mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Make 4% PFA in 1X PBS
- 10ml 16% PFA + 4ml 10X PBS + 26ml H2O (made fresh)
- Take out mouse brain section from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Attach coverslip to bottom of petri dish with ARcare 90106 double sided adhesive
- Add 100ul 0.25% TX-100 in 2XSSPE and incubate 15min at RT
- Wash with cold nf-H2O three times and check for degradation
- Add 100ul 0.01% Pepsin in 0.1N HCl and incubate for X min at 37C
- 3ul 1%Pepsin + 15ul 2N HCl + 282ul H2O
- Sample 1 = 1min, Sample 2 = 5min, Sample 3 = 5min
- Sample 2 was already pretty degraded when I took it out so I also took sample 3 out around 5min mark
- Wash with nf-1X PBS three times and check for degradation
- Sample 2 and 3 could see tissue sloughing off with each wash, doubt anything will be left after RT overnight
- Prepare 400ul Reverse Transcription Mix on ice and add ~133ul to each dish
Components | Volume |
H2O | 159 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM RT primer | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~15hr at 37C
Day 2
- Wash with 1X PBS once
- Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
- Wash with nf-H2O twice
- Add CircLigase mix and incubate 4hr at 60C
- H2O - 128ul
- CircLigase Buffer 10X - 20ul
- MnCl2 50mM - 10ul
- Betaine 5M - 40ul
- CircLigase II 100U/ul - 2ul
- Wash with 1X PBS twice
- Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
- Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
- Prepare RCA reaction mix on ice
- H2O - 174ul
- Phi29 Buffer 10X - 20ul
- dNTP 25mM - 2ul
- aa-dUTP 4mM - 2ul
- Phi29 DNA polymerase 100U/ul - 2ul
- Add RCA mix and incubate at 30C overnight (~15hrs)