Matt:LabNotes/2016-7-14
Jump to navigation
Jump to search
FISSEQ on BA8 sections with PA gel + Acrydite/BS(PEG)9[edit]
- Try FISSEQ to see a "baseline" of number of rolonies
- Gel mix and protocol is same as this with BS(PEG)9
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[edit]
Day 1[edit]
- Prepare a plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Use 4% PFA in 1X PBS from 7/6/2016
- Take out BA8 section 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
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times and check for degradation
- 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 and check for degradation
- Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
- Secure in plastic jig
- Add 50ul gel casting mix filtered and degassed
- 5% gel with 250uM Acrydite-Amine linker and 5mM BS(PEG)9
- Seal in plastic bag and vacuum out air before filling with argon
- Let sit at RT for 30min
- Aspirate non-polymerized gel and wash once with 1X PBS
- Attach coverslip to bottom of petri dish
- Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X SSPE
- 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 FISSEQ_RT primer | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C
Day 2[edit]
- File:20160714 BA8 Hydrogel Day2.JPG
- No swelling
- Good coverage of gel, almost no air bubbles, only edges lack gel which is not a problem
- 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
- Prepare Circligase mix on ice
Component | Volume |
DEPC-H2O | 128 |
CircLigase II Buffer 10X | 20 |
MnCl2 50mM | 10 |
Betaine 5M | 40 |
CircLigase II 100U/ul | 2 |
Total | 200 |
- Add mix to sample and incubate for 3hr at 60C
- 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
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
- Add RCA mix and incubate at 30C overnight (~15hrs)
Day 3[edit]
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 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
Imaging[edit]
- Add 0.5uM FISSEQ_Adpt in 30% formamide + 2X SSC preheated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice
- Image (saved in 7-20-2016)
Results[edit]
- Took images at 6 positions
- Tissue approximately outlined in yellow
File:20160720 BA8 FISSEQ 50umGel Positions.jpg
Position 1[edit]
File:MAX 20160720 BA8 FISSEQ 50umGel Pos1.jpgFile:20160720 BA8 FISSEQ 50umGel Pos1 z00 ch01.jpg
Position 2[edit]
File:MAX 20160720 BA8 FISSEQ 50umGel Pos2.jpgFile:20160720 BA8 FISSEQ 50umGel Pos2 z06 ch01.jpg
Position 3[edit]
File:MAX 20160720 BA8 FISSEQ 50umGel Pos3.jpgFile:20160720 BA8 FISSEQ 50umGel Pos3 z05 ch01.jpg
Position 4[edit]
File:MAX 20160720 BA8 FISSEQ 50umGel Pos4.jpgFile:20160720 BA8 FISSEQ 50umGel Pos4 z03 ch01.jpg
Position 5[edit]
File:MAX 20160720 BA8 FISSEQ 50umGel Pos5.jpgFile:20160720 BA8 FISSEQ 50umGel Pos5 z05 ch01.jpg
Position 6[edit]
File:MAX 20160720 BA8 FISSEQ 50umGel Pos6.jpgFile:20160720 BA8 FISSEQ 50umGel Pos6 z04 ch01.jpg
Conclusion[edit]
- Dropping polyacrylamide mix on coverslip before sandwiching with glass slide works well to prevent bubbles
- Less rolonies than previous FISSEQ on BA8 tissue sections