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
- No additives
- 5% formamide
- 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]
- Prepare 3 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Made 4% PFA in 1X PBS 2 weeks ago stored at RT
- 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
- Take out brain section from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in petri dish 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
- Skip pepsin step usually done here
- 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
- 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 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
- ppCUX2 (dcProbe6-Cy3)
- 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]
- Wash with 1X PBS twice preheated to 55C
- Keep dishes on metal from EZ-oven to maintain temp, let sit ~1min during each wash
- Add SplintR Enzyme and incubate at 37C for 30min
- 20ul 10X Buffer + 6ul SplintR + 174ul H2O (not enough RiboLock for 5ul each)
- Wash with 1X PBS twice, let sit 1min during each wash
- Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 75C and incubate 1hr at 55C
- 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 330ul BS(PEG)9 (20ul BS(PEG)9 + 980ul 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
- Store in PBS at 4C 2 days
FISH[edit]
- Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice
- Image
- Add 80% formamide + 2X SSC preheated to 75C
- Incubate for 15min at RT
- Wash with 1X PBS twice
- Add 80% formamide + 2X SSC preheated to 75C
- Incubate for 15min at RT
- Wash with 1X PBS twice
- Check to see if stripped
- Add 0.5uM dcProbe2-Cy3 (MALAT1) and 0.5uM dcProbe6-488 in 30% formamide + 2X SSC preheated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice
- Image
- Add 0.5uM dcProbe6-Cy3 (CUX2) in 30% formamide + 2X SSC preheated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice
- Image
DRAQ5[edit]
- Aspirate and then add 100ul 5uM DRAQ5 in 1X PBS
- Incubate 30min at RT
- Image