Matt:LabNotes/2015-6-8
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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