Matt:LabNotes/2015-6-8
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RNA in situ Differentiated Motor Neurons
- 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
- Sample: RT 15min
- Labeled "RNAFISH1 6.8.2015"
- Sample: 37C 10min
- Labeled "RNAFISH2 6.8.2015"
- Prepare 8ml 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 110ul hybridization solution to each sample and incubate overnight at 37C (7p-)
Buffer Prep
- Wash Buffer
- 20X SSC 5mL
- Formamide 5mL
- RNase free H2O 40ml
- 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
FISSEQ
- 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 (7pm-)
DARTFISH
- 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 (7pm-)