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=Motor Neurons Third Batch= *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== *[[Matt:LabNotes/2015-6-19 | 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 [[Matt:LabNotes/2015-5-18 | in vitro experiment]] ***e.g. 100nM CA12kNov2014_V4 = 28.5pM per padlock probe, therefore 28.5nM of each suppressor oligo ===DARTFISH with suppv2=== *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 #*[[Matt:JustinLabNotes/2015-7-6 | 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<!-- #Strip with 80% formamide pre-heated to 75C and incubate 15min at RT #Wash with 1X PBS twice and store in 2ml PBS--> ====Imaging==== *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|450px]] *Pos2 [[File:MAX_20150723_DARTFISH_MN_FISGA_Pos2.jpg|450px]] ==sm RNA FISH== *Using [[Matt:LabNotes/2015-7-14 | 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 [[Matt:LabNotes/2015-7-16 | 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=== *[[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]] *[[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]] *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==== *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=== ====Zeiss Microscope in Huang Lab with Laser excitation==== *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) [[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==== *7/22/2015
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