Matt:LabNotes/2016-1-27
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FISSEQ on Mouse Embryo Test 2
- Previous test using sections of fixed embryo
- This time try fresh frozen mouse embryo sections
Embryo Section Info
Protocol
Day 1
- Prepare 2 plastic culture dish with 18mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Use 1 week old 4% PFA at RT
- Take out fresh frozen mouse embryo sections from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 18mm diameter hole (16mm adhesive hole)
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Both dishes leaked and required glue
- Wash with cold nf-H2O three times
- Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with nf-1X PBS three times
- Prepare Reverse Transcription Mix on ice and add
Components | Volume |
H2O | 157 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM FISSEQ_RT | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C
- Parafilm each dish and then put in plastic bag with wet tissue
Day 2
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (10ul BS(PEG)9 stock + 490ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Quench with 1M Tris for 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
- Add 200ul CircLigaseII mix and incuabate 3hr at 60C
Component | Volume |
DEPC-H2O | 640 |
CircLigase Buffer 10X | 100 |
MnCl2 50mM | 50 |
Betaine 5M | 200 |
CircLigase II (100U/ul) | 10 |
Total | 1000 |
- Wash with 1X PBS twice
- Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C
- Wash with 2X SSC + 30% formamide for 10min at 60C
- wash with 2X SSC, 1X SSC, 1X PBS once each
- Add 200ul RCA mix and incubate for 15hr at 30C
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
Day 3
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Quench with 1M Tris for 30min at RT
- Wash with 1X PBS twice