Matt:LabNotes/2015-6-8: Difference between revisions
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>Mzcai m (→RNA FISH) |
>Mzcai m (→Results) |
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(22 intermediate revisions by the same user not shown) | |||
Line 19: | Line 19: | ||
**Labeled "RNAFISH2 6.8.2015" | **Labeled "RNAFISH2 6.8.2015" | ||
#Prepare | #Prepare 10ml Wash Buffer and let sit at RT | ||
#Thaw 500ul aliquot Hybridization Buffer and warm up to 37C | #Thaw 500ul aliquot Hybridization Buffer and warm up to 37C | ||
#*Aliquots made by Dan | #*Aliquots made by Dan | ||
#Prepare hybridization reaction | #Prepare hybridization reaction (2X volume) | ||
#*Hybridization Buffer 200ul | #*Hybridization Buffer 200ul | ||
#*Probe | #*Probe 10ul of each (488 and 594) | ||
#Wash the cells with 1ml of PBS (RNase free) 2 times | #Wash the cells with 1ml of PBS (RNase free) 2 times | ||
#Add hybridization solution to sample and incubate overnight at 37C ( | #Add Wash Buffer and let sit 10min at RT | ||
#Aspirate | |||
#Add 110ul hybridization solution to each sample and incubate overnight at 37C (~18hrs) | |||
#Warm up 6ml Wash Buffer and 2ml Wash Buffer with DAPI to 37C | |||
#Rinse with 2ml Wash Buffer | |||
#Add 1ml Wash Buffer and incubate 30min at 37C | |||
#Replace with 1ml Wash Buffer with DAPI and incubate 30min at 30C | |||
#Wash with 2X SSC (warmed to 30C) twice | |||
#Add 2X SSC | |||
====Buffer Prep==== | ====Buffer Prep==== | ||
Line 33: | Line 41: | ||
**Formamide 5mL | **Formamide 5mL | ||
**RNase free H2O 40ml | **RNase free H2O 40ml | ||
*Wash Buffer with DAPI | |||
**Wash Buffer 10ml | |||
**DAPI (100ug/ml) 50ul | |||
***Stored in brown opaque tube in 4C | |||
*Hybridization Buffer | *Hybridization Buffer | ||
Line 42: | Line 55: | ||
**RVC 200 mM (warm to 37) 100 uL | **RVC 200 mM (warm to 37) 100 uL | ||
**BSA 50 mg/mL 40 uL | **BSA 50 mg/mL 40 uL | ||
====Results==== | |||
*Tried imaging with confocal using 488 and 552 lasers | |||
**Only saw autofluorescence from cell, looked really cool to see all the dendrites/axons branching from cells but no spots | |||
*[https://sites.google.com/site/singlemoleculernafish/faq#TOC-Imaging Arjun Raj FAQ for sm RNA FISH suggests using epifluorescence is better than confocal] | |||
*Used Olympus | |||
**GFP (for Alexa 488) and Texas Red (for Alexa 594) filter cubes | |||
***300ms-1000ms exposure | |||
**DAPI | |||
***~20ms exposure | |||
*Merged the different channels together (blue=DAPI,green=GFP,red=TXRED,grey=BF) | |||
**Green and red overlap everywhere (only yellow can be seen) therefore no specific signal | |||
***Saved in DARTFISH-RNAFISH folder on Desktop (6/10/2015) | |||
***Will try higher expressed genes (KIT and CUX2): [[Matt:LabNotes/2015-6-18#RNA_FISH | Try #2]] | |||
[[File:20150610_MotorNeur_RNAFISH2_Dish5_Pos1_composite.jpg|500px]] | |||
===FISSEQ=== | ===FISSEQ=== | ||
Line 53: | Line 83: | ||
#*dNTP 25mM - 2ul | #*dNTP 25mM - 2ul | ||
#*aa-dUTP 4mM - 2ul | #*aa-dUTP 4mM - 2ul | ||
#* | #*FISSEQ_RT primer 100uM - 5ul | ||
#**/5Phos/TCTCGGGAACGCTGAAGANNNNNN | #**/5Phos/TCTCGGGAACGCTGAAGANNNNNN | ||
#*RNase inhibitor 40U/ul - 2ul | #*RNase inhibitor 40U/ul - 2ul | ||
#*M-MuLV reverse transcriptase 100U/ul - 10ul | #*M-MuLV reverse transcriptase 100U/ul - 10ul | ||
#Add RT mixture and incubate for 10min at 4C | #Add RT mixture and incubate for 10min at 4C | ||
#Transfer sample to 37C overnight ( | #Transfer sample to 37C overnight (~15hrs) | ||
#Wash with 1X PBS once | |||
#Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 1X PBS prepared on ice) to sample and incubate 1hr at RT | |||
#Wash with 1X PBS twice | |||
#Add 200ul 1M Tris (pH 8.0) for 30min at RT | |||
#Wash with 1X PBS twice | |||
#Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C | |||
#Wash with 2ml H2O twice | |||
#Prepare CircLigase mixture '''on ice''' | |||
#*H2O - 128ul | |||
#*CircLigase Buffer 10X - 20ul | |||
#*MnCl2 50mM - 10ul | |||
#*Betaine 5M - 40ul | |||
#*CircLigase II 100U/ul - 2ul | |||
#Add CircLigase mix to sample and incubate 2hr at 60C | |||
#Wash with 1X PBS twice | |||
#Add 200ul (1ul 100uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C | |||
#Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS | |||
#Prepare RCA reaction mix '''on ice''' | |||
#*H2O - 174ul | |||
#*Phi29 Buffer 10X - 20ul | |||
#*dNTP 25mM - 2ul | |||
#*aa-dUTP 4mM - 2ul | |||
#*Phi29 DNA polymerase 100U/ul - 2ul | |||
#Add RCA mix and incubate at 30C overnight (~16 hrs) | |||
#Wash with 1X PBS once | |||
#Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 1X PBS prepared on ice) to sample and incubate 1hr at RT | |||
#Wash with 1X PBS twice | |||
#Add 200ul 1M Tris (pH 8.0) for 30min at RT | |||
#Wash with 1X PBS twice | |||
#Add 0.5uM FISSEQ_Adpt (1ul 100uM FISSEQ_Adpt + 199ul 2XSSC + 30% formamide) pre-heated to 75C and incubate 10min at RT | |||
#Wash with 2X SSC twice and add 1ml 2X SSC for imaging | |||
====Results 6/10/2015==== | |||
*Not many rolonies | |||
Pos1<br> | |||
[[File:Experiment_MotorNeurons_FISSEQ_Pos1_MIP_overlay_BF.jpg|400px]]<br> | |||
Pos2<br> | |||
[[File:Experiment_MotorNeurons_FISSEQ_Pos2_MIP_overlay_BF.jpg|400px]]<br> | |||
Pos3<br> | |||
[[File:Experiment_MotorNeurons_FISSEQ_Pos3_MIP_overlay_BF.jpg|400px]]<br> | |||
===DARTFISH=== | ===DARTFISH=== | ||
Line 77: | Line 147: | ||
#*M-MuLV reverse transcriptase 100U/ul - 20ul | #*M-MuLV reverse transcriptase 100U/ul - 20ul | ||
#Add RT mixture and incubate for 10min at 4C | #Add RT mixture and incubate for 10min at 4C | ||
#Transfer sample to 37C overnight ( | #Transfer sample to 37C overnight (~15hrs) | ||
#Wash with 1X PBS once | |||
#Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 1X PBS prepared on ice) to sample and incubate 1hr at RT | |||
#Wash with 1X PBS twice | |||
#Add 200ul 1M Tris (pH 8.0) for 30min at RT | |||
#Wash with 1X PBS twice | |||
#Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C | |||
#Wash with 2ml H2O twice | |||
#Prepare Ampligase mixture (2X volume) '''on ice''' | |||
#*H2O - 110ul | |||
#*Ampligase Buffer 10X - 20ul | |||
#*[[Matt:LabNotes/2015-5-11#Qubit_Quantification | Padlock Probes 461nM]] - 50ul | |||
#**Heat to 90C first and snap cool on ice block | |||
#*Ampligase 5U/ul - 20ul | |||
#Add Ampligase mix to sample and incubate 3hr at 60C then 21hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed) | |||
#*3:20p-3p | |||
#Wash with 1X PBS once | |||
#Add 200ul (1ul 100uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | |||
#Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS | |||
#Prepare RCA reaction mix (2X volume) '''on ice''' | |||
#*H2O - 348ul | |||
#*Phi29 Buffer 10X - 40ul | |||
#*dNTP 25mM - 4ul | |||
#*aa-dUTP 4mM - 4ul | |||
#*Phi29 DNA polymerase 100U/ul - 4ul | |||
#Add RCA mix and incubate at 30C overnight (~14hrs) | |||
#Wash with 1X PBS once | |||
#Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 1X PBS prepared on ice) to sample and incubate 1hr at RT | |||
#Wash with 1X PBS twice | |||
#Add 200ul 1M Tris (pH 8.0) for 30min at RT | |||
#Wash with 1X PBS twice | |||
#Add 0.5uM FISGA_Adpt (1ul 100uM FISGA_Adpt + 199ul 2XSSC + 30% formamide) pre-heated to 75C and incubate 10min at RT | |||
#Wash with 2X SSC twice and add 1ml 2X SSC for imaging | |||
#Strip with 80% formamide pre-heated to 75C and incubate 15min at RT | |||
#Wash with 1X PBS twice and store in 2ml PBS | |||
====Rolony Check 6/11/2015==== | |||
*Much more rolonies than FISSEQ | |||
*DARTFISH1 | |||
[[File:20150611_MotorNeur_DARTFISH1_Pos1_FISGAadpt_MIP_overlay_BF.jpg|400px]] | |||
*DARTFISH2 | |||
[[File:20150611_MotorNeur_DARTFISH2_Pos1_FISGAadpt_MIP_overlay_BF.jpg|400px]] | |||
====Decoding Results 6/17/2015==== | |||
*[[Matt:LabNotes/2015-6-17]] |
Latest revision as of 21:07, 24 June 2015
RNA in situ Differentiated Motor Neurons[edit]
- Picking up iPS derived motor neurons from Yeo Lab (Sebastian)
- Fixed in 4% paraformaldehyde (fresh) at 8:30a 6/8/2015
- 4 at RT for 10min
- 8 at RT for 15min
- 8 at 37C for 10min
- Permeabilized/store in 70% EtOH at -20C for ~5hr
- After bringing to our lab put in 4C > 3hr
- Fixed in 4% paraformaldehyde (fresh) at 8:30a 6/8/2015
RNA FISH[edit]
- Sample: RT 15min
- Labeled "RNAFISH1 6.8.2015"
- Sample: 37C 10min
- Labeled "RNAFISH2 6.8.2015"
- Prepare 10ml Wash Buffer and let sit at RT
- Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
- Aliquots made by Dan
- Prepare hybridization reaction (2X volume)
- Hybridization Buffer 200ul
- Probe 10ul of each (488 and 594)
- Wash the cells with 1ml of PBS (RNase free) 2 times
- Add Wash Buffer and let sit 10min at RT
- Aspirate
- Add 110ul hybridization solution to each sample and incubate overnight at 37C (~18hrs)
- Warm up 6ml Wash Buffer and 2ml Wash Buffer with DAPI to 37C
- Rinse with 2ml Wash Buffer
- Add 1ml Wash Buffer and incubate 30min at 37C
- Replace with 1ml Wash Buffer with DAPI and incubate 30min at 30C
- Wash with 2X SSC (warmed to 30C) twice
- Add 2X SSC
Buffer Prep[edit]
- Wash Buffer
- 20X SSC 5mL
- Formamide 5mL
- RNase free H2O 40ml
- Wash Buffer with DAPI
- Wash Buffer 10ml
- DAPI (100ug/ml) 50ul
- Stored in brown opaque tube in 4C
- Hybridization Buffer
- RNAse free water 5.3 mL
- SSC 20X 1 mL
- Dextran sulfate 2 mL
- Formamide 1 mL
- E coli tRNA 500 uL
- RVC 200 mM (warm to 37) 100 uL
- BSA 50 mg/mL 40 uL
Results[edit]
- Tried imaging with confocal using 488 and 552 lasers
- Only saw autofluorescence from cell, looked really cool to see all the dendrites/axons branching from cells but no spots
- Arjun Raj FAQ for sm RNA FISH suggests using epifluorescence is better than confocal
- Used Olympus
- GFP (for Alexa 488) and Texas Red (for Alexa 594) filter cubes
- 300ms-1000ms exposure
- DAPI
- ~20ms exposure
- GFP (for Alexa 488) and Texas Red (for Alexa 594) filter cubes
- Merged the different channels together (blue=DAPI,green=GFP,red=TXRED,grey=BF)
- Green and red overlap everywhere (only yellow can be seen) therefore no specific signal
- Saved in DARTFISH-RNAFISH folder on Desktop (6/10/2015)
- Will try higher expressed genes (KIT and CUX2): Try #2
- Green and red overlap everywhere (only yellow can be seen) therefore no specific signal
File:20150610 MotorNeur RNAFISH2 Dish5 Pos1 composite.jpg
FISSEQ[edit]
- Sample: RT 15min
- Labeled "FISSEQ +ctrl 6.8.2015"
- Wash the cells with 1ml of PBS (RNase free) 3 times
- Prepare RT mixture on ice
- DEPC-H2O - 159ul
- M-MuLV RT Buffer 10X - 20ul
- dNTP 25mM - 2ul
- aa-dUTP 4mM - 2ul
- FISSEQ_RT primer 100uM - 5ul
- /5Phos/TCTCGGGAACGCTGAAGANNNNNN
- RNase inhibitor 40U/ul - 2ul
- M-MuLV reverse transcriptase 100U/ul - 10ul
- Add RT mixture and incubate for 10min at 4C
- Transfer sample to 37C overnight (~15hrs)
- Wash with 1X PBS once
- Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 200ul 1M Tris (pH 8.0) for 30min at RT
- Wash with 1X PBS twice
- Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
- Wash with 2ml H2O twice
- Prepare CircLigase mixture on ice
- H2O - 128ul
- CircLigase Buffer 10X - 20ul
- MnCl2 50mM - 10ul
- Betaine 5M - 40ul
- CircLigase II 100U/ul - 2ul
- Add CircLigase mix to sample and incubate 2hr at 60C
- Wash with 1X PBS twice
- Add 200ul (1ul 100uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
- Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
- Prepare RCA reaction mix on ice
- H2O - 174ul
- Phi29 Buffer 10X - 20ul
- dNTP 25mM - 2ul
- aa-dUTP 4mM - 2ul
- Phi29 DNA polymerase 100U/ul - 2ul
- Add RCA mix and incubate at 30C overnight (~16 hrs)
- Wash with 1X PBS once
- Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 200ul 1M Tris (pH 8.0) for 30min at RT
- Wash with 1X PBS twice
- Add 0.5uM FISSEQ_Adpt (1ul 100uM FISSEQ_Adpt + 199ul 2XSSC + 30% formamide) pre-heated to 75C and incubate 10min at RT
- Wash with 2X SSC twice and add 1ml 2X SSC for imaging
Results 6/10/2015[edit]
- Not many rolonies
Pos1
File:Experiment MotorNeurons FISSEQ Pos1 MIP overlay BF.jpg
Pos2
File:Experiment MotorNeurons FISSEQ Pos2 MIP overlay BF.jpg
Pos3
File:Experiment MotorNeurons FISSEQ Pos3 MIP overlay BF.jpg
DARTFISH[edit]
- Sample: RT 15min
- Labeled "DARTFISH1 6.8.2015"
- Sample: 37C 10min
- Labeled "DARTFISH2 6.8.2015"
- Wash the cells with 1ml of PBS (RNase free) 3 times
- Prepare RT mixture on ice
- DEPC-H2O - 318ul
- M-MuLV RT Buffer 10X - 40ul
- dNTP 25mM - 4ul
- aa-dUTP 4mM - 4ul
- RT primer 100uM - 10ul
- Nonamer
- RNase inhibitor 40U/ul - 4ul
- M-MuLV reverse transcriptase 100U/ul - 20ul
- Add RT mixture and incubate for 10min at 4C
- Transfer sample to 37C overnight (~15hrs)
- Wash with 1X PBS once
- Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 200ul 1M Tris (pH 8.0) for 30min at RT
- Wash with 1X PBS twice
- Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
- Wash with 2ml H2O twice
- Prepare Ampligase mixture (2X volume) on ice
- H2O - 110ul
- Ampligase Buffer 10X - 20ul
- Padlock Probes 461nM - 50ul
- Heat to 90C first and snap cool on ice block
- Ampligase 5U/ul - 20ul
- Add Ampligase mix to sample and incubate 3hr at 60C then 21hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed)
- 3:20p-3p
- Wash with 1X PBS once
- Add 200ul (1ul 100uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
- Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
- Prepare RCA reaction mix (2X volume) on ice
- H2O - 348ul
- Phi29 Buffer 10X - 40ul
- dNTP 25mM - 4ul
- aa-dUTP 4mM - 4ul
- Phi29 DNA polymerase 100U/ul - 4ul
- Add RCA mix and incubate at 30C overnight (~14hrs)
- Wash with 1X PBS once
- Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 200ul 1M Tris (pH 8.0) for 30min at RT
- Wash with 1X PBS twice
- Add 0.5uM FISGA_Adpt (1ul 100uM FISGA_Adpt + 199ul 2XSSC + 30% formamide) pre-heated to 75C and incubate 10min at RT
- Wash with 2X SSC twice and add 1ml 2X SSC for imaging
- Strip with 80% formamide pre-heated to 75C and incubate 15min at RT
- Wash with 1X PBS twice and store in 2ml PBS
Rolony Check 6/11/2015[edit]
- Much more rolonies than FISSEQ
- DARTFISH1
File:20150611 MotorNeur DARTFISH1 Pos1 FISGAadpt MIP overlay BF.jpg
- DARTFISH2
File:20150611 MotorNeur DARTFISH2 Pos1 FISGAadpt MIP overlay BF.jpg