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==Motor Neurons DARTFISH CA12kNov14_suppv2== *[[Matt:LabNotes/2015-6-18 | Picked up second batch iPS derived motor neurons from Yeo Lab (Sebastian)]] **Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015 ***RT for 15min **Permeabilized/store in 70% EtOH at 4C (~72 hours before this DARTFISH experiment) *[[Matt:LabNotes/2015-6-8#DARTFISH | Last time forgot to add suppressor oligos]] **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 6.19.2015" #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 50nM (labeled supp V4 ALL 5.18.15) - 55ul #*Ampligase Buffer 10X - 10ul #*[[Matt:LabNotes/2015-6-3#Qubit_ssDNA_Quantification | Padlock Probes 438nM]] - 25ul #**Heat to 90C first and snap cool on ice block #*Ampligase 5U/ul - 10ul #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) #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 (~14hrs) #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=== *Continued onn [[Matt:LabNotes/2015-7-9]]
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