Matt:LabNotes/2015-7-20
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Motor Neurons Third Batch[edit]
- Picked up third batch iPS derived motor neurons from Yeo Lab (Sebastian) fixed on 7/17/2015 at 5pm
- Fixed in 4% paraformaldehyde RT for 15min
- Permeabilized/store in 70% EtOH at 4C (~72 hours before these experiments)
DARTFISH CA12kNov14_suppv2[edit]
- Repeat of second batch DARTFISH CA12kNov14_suppv2
- Teach Justin how to do DARTFISH
- May not decode these samples unless successful RNA FISH on this batch
- Use 1000X concentration of suppressor oligos to match in vitro experiment
- e.g. 100nM CA12kNov2014_V4 = 28.5pM per padlock probe, therefore 28.5nM of each suppressor oligo
DARTFISH with suppv2[edit]
- Sample labeled "DARTFISH 7.20.2015"
- Wash the cells with 1ml of PBS (RNase free) 3 times
- Prepare RT mixture on ice
- DEPC-H2O - 157ul
- M-MuLV RT Buffer 10X - 20ul
- dNTP 25mM - 2ul
- aa-dUTP 2mM - 4ul
- RT primer 100uM - 5ul
- Nonamer
- 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 (~18hrs)
- Wash with 1X PBS once
- Add 300ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 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 on ice
- Suppressor Oligos 100nM (labeled supp V4 ALL 5.18.15) - 25.9ul
- Ampligase Buffer 10X - 9.1ul
- Padlock Probes 239nM - 38ul
- Heat to 90C first and snap cool on ice block
- Ampligase 5U/ul - 9.1ul
- H2O - 7.9ul
- Total: 91ul
- Add Ampligase mix to sample and incubate 3hr at 60C then 25hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed)
- Incubated long time due to timing of starting RCA
- Middle of glass looked almost dry but edges were clearly wet
- Wash with 1X PBS once
- Add 200ul (1ul 200uM 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 on ice
- H2O - 172ul
- Phi29 Buffer 10X - 20ul
- dNTP 25mM - 2ul
- aa-dUTP 2mM - 4ul
- Phi29 DNA polymerase 100U/ul - 2ul
- Add RCA mix and incubate at 30C overnight (~16hrs)
- Wash with 1X PBS once
- Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 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
Imaging[edit]
- Check with Universal Rolony FISGA_Adpt
- ~30 rolonies per cell
- Not great, could be better
- High cell density
- Pos1
File:MAX 20150723 DARTFISH MN FISGA Pos1.jpg
- Pos2
File:MAX 20150723 DARTFISH MN FISGA Pos2.jpg
sm RNA FISH[edit]
- Using KIT-488 CUX2-594
- Expect to see very few to no signals in FITC channel due to few KIT mRNA
- If TXRED channel shows CUX2 transcripts then dye:probe=1.3 is not the problem
- If TXRED channel shows bright regions then probe sequences not the problem
- Using SNAP25-488 CUX2-647
- Expect to see many SNAP25 spots in FITC channel
- Hopefully can also see a couple CUX2 spots in Cy5 channel
Procedure[edit]
- Sample 1 labeled: "RNAFISH1 7.20.2015"
- KIT-488 CUX2-594
- Sample 2 labeled: "RNAFISH2 7.20.2015"
- SNAP25-488 CUX2-647
- Prepare 10ml Wash Buffer and let sit at RT
- Thaw 300ul aliquot Hybridization Buffer and warm up to 37C
- Aliquots made by Dan
- Prepare hybridization reaction
- Hybridization Buffer 100ul
- Probe 5ul + 5ul
- Wash the cells with 1ml of PBS (RNase free) 2 times
- Add 2ml Wash Buffer and let sit 10min at RT
- Aspirate
- Add 110ul hybridization solution to each sample and incubate overnight at 37C (~23hrs)
- Prepare 12ml Wash Buffer
- Warm up 8ml Wash Buffer and 4ml Wash Buffer with DAPI to 37C
- Rinse with 2ml Wash Buffer
- Add 2ml Wash Buffer and incubate 30min at 37C
- Replace with 2ml 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
Imaging[edit]
Zeiss Microscope in Huang Lab with Laser excitation[edit]
- 7/21/2015
- With help of Matt Walsh
- Laser wavelengths:
- 488
- 552?
- 642
- Exposure time: 100ms
- Sample 1 - 488 (KIT)
- Nothing to show, probably because gene expression is too low
- Sample 1 - 594 (CUX2)
- See combination of bright regions (guess is due to uncoupled dyes) and very few bright spots
File:20150720 RNAFISH1 594.jpg
- Sample 2 - 488 (SNAP25)
- Lots of spots but also high background
- Hard to say significantly more than what I have seen with CUX2 (though based on DARTFISH and FPKM it should be)
- Lots of spots but also high background
File:20150720 RNAFISH2 488.jpg
- Sample 2 - 647 (CUX2)
- Image is very grainy making it hard to tell distinct spots from noise, probably needs better excitation parameters
File:20150720 RNAFISH2 647.jpg
Zeiss Microscope in Huang Lab with Metal-Halide light[edit]
- 7/22/2015