Matt:LabNotes/2015-8-18: Difference between revisions
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>Mzcai (Created page with "=BA8 Section DARTFISH suppv2 with Fiducial Beads= *Similar to Matt:LabNotes/2015-8-4 but with suppressor oligos and 5 cycle decoding *3-4 month old BA8 tissue section *su...") |
>Mzcai |
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=BA8 Section DARTFISH suppv2 with Fiducial Beads= | =BA8 Section DARTFISH suppv2 with Fiducial Beads= | ||
* | *Repeat [[Matt:LabNotes/2015-8-17]] but with fresh PFA and 0.01N HCl (instead of 0.1N during pepsin step) to see if that helps with tissue degradation | ||
*3-4 month old BA8 tissue section | *3-4 month old BA8 tissue section | ||
*suppv2 design | *suppv2 design | ||
*Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers | *Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers | ||
*Try only 5min incubation with TX-500 to see if that decreases tissue degradation | *Try only 5min incubation with TX-500 to see if that decreases tissue degradation | ||
==DARTFISH on BA8== | ==DARTFISH on BA8== | ||
#Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate | #Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate | ||
#*EtOH sterilize and UV | #*EtOH sterilize and UV | ||
# | #Made 40ml 4% PFA at RT | ||
#*10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O | |||
#Take out BA8 section from -80C and incubate on 50C hot plate for 3min | #Take out BA8 section from -80C and incubate on 50C hot plate for 3min | ||
#Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C | #Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C | ||
#Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE | #Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE | ||
#Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive | #Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive | ||
#Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT | #Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT | ||
#*New solution made by Justin | #*New solution made by Justin | ||
#Wash with nuclease-free H2O three times | #Wash with nuclease-free H2O three times | ||
#Add 0.01% Pepsin in 0. | #Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C | ||
#*2ul 1%Pepsin + | #*2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O | ||
#Wash with nuclease-free PBS three times | #Wash with nuclease-free PBS three times | ||
#Prepare Reverse Transcription Mix '''on ice''' | #Prepare Reverse Transcription Mix '''on ice''' | ||
Line 53: | Line 52: | ||
#RNA Removal | #RNA Removal | ||
#*Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C | #*Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C | ||
#Wash with H2O twice | |||
#Wash with | #Add 100nM Padlock Probe Mix with 1000X suppv2 oligos and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr | ||
#*Wrapped with parafilm to prevent evaporation | |||
#Add | |||
#*Wrapped with parafilm to prevent evaporation | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| DEPC-H2O|| | | DEPC-H2O||20.18 | ||
|- | |- | ||
| Ampligase Buffer|| | | Ampligase Buffer||10 | ||
|- | |- | ||
| [[Matt:JustinLabNotes/2015-7- | | [[Matt:JustinLabNotes/2015-7-22#Qubit_Quantification | Padlock Probes 320nM]]||31.25 | ||
|- | |- | ||
| | | 100nM suppv2 Oligos || 28.57 | ||
|- | |- | ||
| Total|| | | Ampligase||10 | ||
|- | |||
| Total||100 | |||
|} | |} | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 1uM FISSEQ_ppRCA ( | #Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | ||
#*Preheated to | #*Preheated to 70C before adding | ||
#Wash with 2X SSC once, 1X SSC once, and 1X PBS once | #Wash with 2X SSC once, 1X SSC once, and 1X PBS once | ||
#Add RCA mix and incubate 6hr at 30C | #Add RCA mix and incubate 6hr at 30C | ||
Line 96: | Line 94: | ||
#Wash with 10mM HEPES | #Wash with 10mM HEPES | ||
# | #Sonicate [[Matt:LabNotes/2015-8-13#Add_Beads | 1:2000 diluted beads]] for 3 minutes in [https://contenti.com/l-r-pc3-compact-ultrasonic-cleaner sonicator bath] | ||
# | #Add 200ul to sample and incubate 5min at RT | ||
#Add to sample and incubate 5min at RT | |||
#Wash with 10mM HEPES once | #Wash with 10mM HEPES once | ||
#Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) and incubate at RT for 1hr | #Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) and incubate at RT for 1hr | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
#Add 1M Tris pH 8.0 and incubate at RT for 30min | #Add 1M Tris pH 8.0 and incubate at RT for 30min | ||
#Wash with 1X PBS twice and store in 1X PBS at 4C overnight- | #Wash with 1X PBS twice and store in 1X PBS at 4C overnight | ||
==Decoding== | |||
*Cycle00 = Fiducial beads (488) and FISGA_Adpt (Cy3) | |||
*Cycle01 = dcProbe1 | |||
*Cycle02 = dcProbe2 | |||
*Cycle03 = dcProbe3 | |||
*Cycle04 = dcProbe4 | |||
*Cycle05 = dcProbe5 | |||
*Cycle06 = Repeat of Cycle01 | |||
**When aspirating there was little solution on the dish... | |||
***did I forget to wash with 2X SSC before starting imaging? | |||
***Is the room so hot that it evaporated in an hour? | |||
*63X objective (184um x 184um FOV) | |||
*z step size = 0.3 um | |||
*z stack size = 50-51 zSlices | |||
*6 Positions | |||
**Each position takes ~11min to image | |||
**Each position takes ~10GB and is taxing on memory |
Latest revision as of 20:45, 29 September 2015
BA8 Section DARTFISH suppv2 with Fiducial Beads[edit]
- Repeat Matt:LabNotes/2015-8-17 but with fresh PFA and 0.01N HCl (instead of 0.1N during pepsin step) to see if that helps with tissue degradation
- 3-4 month old BA8 tissue section
- suppv2 design
- Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers
- Try only 5min incubation with TX-500 to see if that decreases tissue degradation
DARTFISH on BA8[edit]
- Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
- EtOH sterilize and UV
- Made 40ml 4% PFA at RT
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
- Take out BA8 section from -80C and incubate on 50C hot plate for 3min
- Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
- New solution made by Justin
- Wash with nuclease-free H2O three times
- Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
- Wash with nuclease-free PBS three times
- Prepare Reverse Transcription Mix on ice
Components | Volume |
H2O | 157 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
100uM N9 | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C
- Wash with 1X PBS once
- Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Add Tris pH8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
- Wash with H2O twice
- Add 100nM Padlock Probe Mix with 1000X suppv2 oligos and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr
- Wrapped with parafilm to prevent evaporation
Component | Volume |
DEPC-H2O | 20.18 |
Ampligase Buffer | 10 |
Padlock Probes 320nM | 31.25 |
100nM suppv2 Oligos | 28.57 |
Ampligase | 10 |
Total | 100 |
- Wash with 1X PBS once
- Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
- Preheated to 70C before adding
- Wash with 2X SSC once, 1X SSC once, and 1X PBS once
- Add RCA mix and incubate 6hr at 30C
Component | Volume |
H2O | 172 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
Phi 29 (low conc) | 2 |
Total | 200 |
- Wash with 10mM HEPES
- Sonicate 1:2000 diluted beads for 3 minutes in sonicator bath
- Add 200ul to sample and incubate 5min at RT
- Wash with 10mM HEPES once
- Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) 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 and store in 1X PBS at 4C overnight
Decoding[edit]
- Cycle00 = Fiducial beads (488) and FISGA_Adpt (Cy3)
- Cycle01 = dcProbe1
- Cycle02 = dcProbe2
- Cycle03 = dcProbe3
- Cycle04 = dcProbe4
- Cycle05 = dcProbe5
- Cycle06 = Repeat of Cycle01
- When aspirating there was little solution on the dish...
- did I forget to wash with 2X SSC before starting imaging?
- Is the room so hot that it evaporated in an hour?
- When aspirating there was little solution on the dish...
- 63X objective (184um x 184um FOV)
- z step size = 0.3 um
- z stack size = 50-51 zSlices
- 6 Positions
- Each position takes ~11min to image
- Each position takes ~10GB and is taxing on memory