Matt:LabNotes/2015-8-18: Difference between revisions

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=BA8 Section DARTFISH suppv2 with Fiducial Beads=
=BA8 Section DARTFISH suppv2 with Fiducial Beads=
*Similar to [[Matt:LabNotes/2015-8-4]] but with suppressor oligos and 5 cycle decoding
*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
*PFA frozen from [[Matt:LabNotes/2015-8-4]]
*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
#Thaw 5ml 4% PFA at RT
#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
#*Still had leak so used gap-filling adhesive 200 to seal
#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 0.1N HCl and incubate for 5min at 37C
#Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C
#*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
#*2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
#*There was '''significant''' degradation after this step, only a sliver of tissue left
#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'''
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#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
#*Even the sliver of tissue has lost more
#Wash with H2O twice
#Wash with 1X PBS twice and then store in 1X PBS parafilmed
#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
#*Put in 4C box labeled "RNA FISH and DARTFISH Cultured Neurons"
#*Wrapped with parafilm to prevent evaporation
<!--
#Add 66nM Ampligase Mix (no suppressor) and incubate ~3hr at 60C as oven slowly decreases to 55C and held for another ~16hr
#*Wrapped with parafilm to prevent evaporation (works very well)
{| {{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||121
| DEPC-H2O||20.18
|-
|-
| Ampligase Buffer||20
| Ampligase Buffer||10
|-
|-
| [[Matt:JustinLabNotes/2015-7-9#Qubit_Quantification | Padlock Probes 337nM]]||39
| [[Matt:JustinLabNotes/2015-7-22#Qubit_Quantification | Padlock Probes 320nM]]||31.25
|-
|-
| Ampligase||20
| 100nM suppv2 Oligos || 28.57
|-
|-
| Total||200
| Ampligase||10
|-
| Total||100
|}
|}
#Wash with 1X PBS once
#Wash with 1X PBS once
#Add 1uM FISSEQ_ppRCA (0.5ul 200uM FISSEQ_ppRCA + 199.5ul 2X SSC + 30% formamide) and incubate 1hr at 55C
#Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
#*Preheated to 65C before adding
#*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
#Dilute beads 1:500 in 10mM HEPES (1ul stock beads + 249ul H2O + 250ul 20mM HEPES pH 7.3)
#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]
#*Make 1.2ml aliquots of stock beads and store in dcProbe box in 4C
#Add 200ul to sample and incubate 5min at RT
#Sonicate for 3 minutes with microtip probe in 1.5ml tube
#*Power = 3, 3sec on 2sec off
#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]

  1. Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH sterilize and UV
  2. Made 40ml 4% PFA at RT
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
  3. Take out BA8 section from -80C and incubate on 50C hot plate for 3min
  4. Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
    • New solution made by Justin
  8. Wash with nuclease-free H2O three times
  9. Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
  10. Wash with nuclease-free PBS three times
  11. 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
  1. Incubate 10min at 4C and then ~18hr at 37C
  2. Wash with 1X PBS once
  3. Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT
  4. Wash with 1X PBS twice
  5. Add Tris pH8.0 and incubate 30min at RT
  6. Wash with 1X PBS twice
  7. RNA Removal
    • Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  8. Wash with H2O twice
  9. 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
  1. Wash with 1X PBS once
  2. Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 70C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. 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
  1. Wash with 10mM HEPES
  2. Sonicate 1:2000 diluted beads for 3 minutes in sonicator bath
  3. Add 200ul to sample and incubate 5min at RT
  4. Wash with 10mM HEPES once
  5. Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) and incubate at RT for 1hr
  6. Wash with 1X PBS twice
  7. Add 1M Tris pH 8.0 and incubate at RT for 30min
  8. 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?
  • 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