Matt:LabNotes/2016-11-29
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DARTFISH on 5342 Occipital Cortex with SplintR in PA Gel[edit]
- Try 1 sample with polyacrylamide gel added after permeabilization
- Used 100nM padlock probe (mix made previously)
- Sample: GTEx Patient 5342 Occipital Cortex sectioned by Yun on 11-22-2016
- 5% Gel mix and protocol is same as this with Acryloyl-X
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]
- Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Made 4% PFA in 1X PBS
- 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
- Prepared SplintR Mix previously
- Preheat Padlock Probes then snap cool
Component | Volume |
10X SplintR Buffer | 10 |
CA12kOct2016_V4 160nM | 60 |
H2O | 30 |
Total | 100 |
- Move sample to 60C HybEZ oven and slowly decreases to 55C and held for ~24hr
Day 2[edit]
- Wash with 1X PBS twice preheated to 55C
- Add SplintR Enzyme and incubate at 37C for 20min
- 20ul 10X Buffer + 6ul SplintR + 174ul H2O
- Wash with 1X PBS twice
- Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C 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 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 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
- Sonicate 1:4000 diluted fluorescent spheres in bath sonicator
- Wash sample with 10mM HEPES
- Add beads to sample and incubate 5min at RT
- Wash sample with 10mM HEPES twice
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
- Wash with 1X PBS twice
- Add 1X Tris pH 8.0 for 30min at RT
- Wash with 1X PBS twice
Imaging[edit]
- Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice