Matt:LabNotes/2016-7-14

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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]

  1. Prepare a plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Use 4% PFA in 1X PBS from 7/6/2016
  3. Take out BA8 section from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  7. Wash with cold nf-H2O three times and check for degradation
  8. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  9. Wash with nf-1X PBS three times and check for degradation
  10. Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
    • Secure in plastic jig
  11. Add 50ul gel casting mix filtered and degassed
    • 5% gel with 250uM Acrydite-Amine linker and 5mM BS(PEG)9
  12. Seal in plastic bag and vacuum out air before filling with argon
  13. Let sit at RT for 30min
  14. Aspirate non-polymerized gel and wash once with 1X PBS
  15. Attach coverslip to bottom of petri dish
  16. Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X SSPE
  17. 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
  1. Incubate 10min at 4C and then ~18hr at 37C

Day 2[edit]

  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice
  6. RNA Removal
    • Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  7. Wash with nf-H2O twice
  8. 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
  1. Add mix to sample and incubate for 3hr at 60C
  2. Wash with 1X PBS twice
  3. Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
  4. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  5. 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
  1. Add RCA mix and incubate at 30C overnight (~15hrs)

Day 3[edit]

  1. Wash with 1X PBS once
  2. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 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

Imaging[edit]

  1. Add 0.5uM FISSEQ_Adpt in 30% formamide + 2X SSC preheated to 75C
  2. Incubate for 10min at RT
  3. Wash with 2X SSC twice
  4. 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