Matt:LabNotes/2016-9-21

From ZhangLabWiki
Jump to navigation Jump to search

DARTFISH on BA8 with SplintR in PA Gel[edit]

  • Try 1 sample with polyacrylamide gel added after permeabilization
  • Used 200nM padlock probe (twice the normal concentration)
Component Volume
40% AB 199:1 Mix 6.25
10% BSA 1
10mg/ml Acryloyl-X, SE in DMSO 1
H2O 39.75
5% TEMED 1
5% APS 1
Total 50

Protocol[edit]

Day 1[edit]

  1. Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Made 4% PFA in 1X PBS on 8/31/2016
    • 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
  3. Take out BA8 section from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in petri dish 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
  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 1X PBS
  17. Prepare SplintR Mix
    • Preheat Padlock Probes then snap cool
Component Volume
10X SplintR Buffer 10
268nM 80
H2O 10
Total 100
  1. Move sample to 60C and oven slowly decreases to 55C and held for ~24hr

Day 2[edit]

  1. Wash with 1X PBS twice preheated to 55C
    • Sample was dry in center, unusual amount of condensation on lid. Image borders of tissue should be okay.
  2. Add SplintR Enzyme and incubate at 37C for 20min
    • 20ul 10X Buffer + 6ul SplintR + 174ul H2O
  3. Wash with 1X PBS twice
  4. Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C
  5. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  6. 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 FISGA_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 9-23-2016)
    • Taken with zstep = 1um except 'Edge' with zstep = 0.3um
  5. 3 Images taken
    • Middle - the area that dried up during padlock probe capture
    • Edge - Close to superficial layer that most likely did not dry
    • Between - Somewhere in between middle and edge

Results[edit]

Middle[edit]

File:MAX Middle.jpg

Between[edit]

File:MAX Between.jpg

Edge[edit]

File:MAX Edge.jpg

Conclusion[edit]

  • Rolony density is higher than ever seen before with FISSEQ or DARTFISH
    • Especially in 'edge' image, which is near superficial layer 1 of neocortex that normally has least amount of rolonies
  • Specificity probably good because of nuclear enrichment of rolonies
    • If non-specific then wouldn't see so much more in nucleus than anywhere else
  • Decreased rolony density in middle is most likely due to dried up during padlock probe annealing and not indicative of poor results
  • NEXT STEP: Decode this sample to prove real rolonies and then Order New Padlock Probes that is RevComp of TB12k_Apr2016