Matt:LabNotes/2017-7-6

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DARTFISH SplintR with 5% formamide or 10% DMF: 3 Probes[edit]

Samples[edit]

  • 10um sections cut by Yun on 4/11/2017
  • 3 samples
    1. No additives
    2. 5% formamide
    3. 10% dimethylformamide
  • 5% Gel mix with 1% crosslinker (normally 0.5%) and protocol is same as this with Acryloyl-X
Component Volume
40% AB 99:1 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 3 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 2 weeks ago stored at RT
    • 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
  3. Take out brain 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. Skip pepsin step usually done here
  9. Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
    • Secure in plastic jig
  10. Add 50ul gel casting mix filtered and degassed
  11. Seal in plastic bag and vacuum out air before filling with argon
  12. Let sit at RT for 30min
  13. Aspirate non-polymerized gel and wash once with 1X PBS
  14. Attach coverslip to bottom of petri dish
  15. Aspirate non-polymerized gel and wash once with 1X PBS

Prepare Padlock Probes[edit]

  • 1ul 10uM (50nM) each padlock probe
    • ppCUX2 (dcProbe6-Cy3)
      • /5Phos/TTCACCCTCTTGGTGATGGAGTAC CTTCAGCTTCCCGATATC TCGTAACCCGTGCGAAGTGC CTAGATTGTTGTCTGTGAGGACCTCC
    • ppBCL11B (dcProbe6-488)
      • /5Phos/AGCAGCTCAGTTTGCATTGCTT CTTCAGCTTCCCGATATC CACGCTTACGATCCCGCTAT TTGTTTCACAATTTCTCAAGGACTAA
    • ppMALAT1 (dcProbe2-Cy3)
      • /5Phos/TTTCTGCCTTTACTTATCAATTC CTTCAGCTTCCCGATATC CGACGGT CTACTTCGTCGCGTCAGACC AAATGGAGGTATGACATATAATCT
  • 20ul 10X (1X) SplintR Buffer
  • 10ul (5%) formamide or 20ul (10%) DMF or 0ul
  • 162ul H2O, 152ul H2O, or 172ul H2O
  • Pre-heat to 95C and then add to dish
  • Add 5ul 40U/ul (1U/ul) RNase Inhibitor
  • Put at 60C in EZ oven and set to 55C to incubate overnight
    • Forgot to set to 55C so did 60C overnight and then 4.5hrs at 55C

Day 2[edit]

  1. Wash with 1X PBS twice preheated to 55C
    • Keep dishes on metal from EZ-oven to maintain temp, let sit ~1min during each wash
  2. Add SplintR Enzyme and incubate at 37C for 30min
    • 20ul 10X Buffer + 6ul SplintR + 174ul H2O (not enough RiboLock for 5ul each)
  3. Wash with 1X PBS twice, let sit 1min during each wash
  4. Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 75C 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 330ul BS(PEG)9 (20ul BS(PEG)9 + 980ul 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
  6. Store in PBS at 4C 2 days

FISH[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
  5. Add 80% formamide + 2X SSC preheated to 75C
  6. Incubate for 15min at RT
  7. Wash with 1X PBS twice
  8. Add 80% formamide + 2X SSC preheated to 75C
  9. Incubate for 15min at RT
  10. Wash with 1X PBS twice
  11. Check to see if stripped
  12. Add 0.5uM dcProbe2-Cy3 (MALAT1) and 0.5uM dcProbe6-488 in 30% formamide + 2X SSC preheated to 75C
  13. Incubate for 10min at RT
  14. Wash with 2X SSC twice
  15. Image
  16. Add 0.5uM dcProbe6-Cy3 (CUX2) in 30% formamide + 2X SSC preheated to 75C
  17. Incubate for 10min at RT
  18. Wash with 2X SSC twice
  19. Image

DRAQ5[edit]

  1. Aspirate and then add 100ul 5uM DRAQ5 in 1X PBS
  2. Incubate 30min at RT
  3. Image