Matt:LabNotes/2015-10-7

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Revision as of 01:47, 8 October 2015 by >Mzcai (→‎FISSEQ on BA8 for Harvard)
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FISSEQ on Mouse Embryo Test

  • Try 2 samples: A5 (20um thickness) and A6 (26um thickness)

Protocol

Day 1

  1. Sterilize culture dishes and tweezers with EtOH, RNaseZap, and then rinse with nf-H2O
  2. Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 16mm diameter hole
    • Use Double-sided tape: Adhesives Research IS-7876-35
  3. Wash tissue section twice using cold nf-H2O for 5min each
  4. Add 0.25% TX-100 in nf-H2O for 5min at RT
  5. Wash with cold nf-H2O twice
  6. Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O) for 10min at RT
  7. Wash with 2ml of cold nf-PBS three times
  8. Prepare 2X Reverse Transcription Mix on ice
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
  1. Incubate 10min at 4C and then ~18hr at 37C
    • Parafilm each dish and then put in plastic bag with wet tissue

FISSEQ on BA8 for Harvard

  • 1 slide with 2 sections and 1 coverslip

Day 1

  1. Prepare 1 plastic culture dishes with hole, tweezers, large plastic dish, slide gasket, and 50C hot plate
    • EtOH and RNaseZap sterilize and rinse with nf-H2O
  2. Made 40ml 4% PFA at RT
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nf-H2O
  3. Take out BA8 section (1 glass slide1 and 1 coverslip1) from -80C and incubate on 50C hot plate for 3min
  4. Submerge coverslip1 in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Submerge glass slides in 35ml 4%PFA in 50ml tube for 15min at 37C
  6. Wash with 2X SSPE by submerging
  7. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  8. Aspirate any liquid and attach gaskets to glass slides
  9. Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
  10. Wash with nuclease-free H2O three times
  11. Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
  12. Wash with nuclease-free PBS three times
  13. Prepare 3X Reverse Transcription Mix on ice
Components Volume
H2O 477
10X M-MuLV Buffer 60
25mM dNTP 6
4mM aa-dUTP 6
100uM N9 or 100uM FISSEQ_RT 15
RNase Inhibitor 6
M-MuLV RTase 30
Total 600
  1. Incubate 10min at 4C and then ~18hr at 37C
    • Put slide in pipette tip box with bottom filled with water and parafilmed around edges of box
    • Parafilm dish and put in humidified bag with mouse E7.5