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===Gene decoding with Barcoded Padlock probes=== ====Experimental plan==== *Hybridizing padlock probes on 1st rolonies, and *Generate 2nd rolonies over 1st rolonies, and *Observe each different fluorescent signal from 2nd rolonies ====Procedure==== *Prepare two barcoded padlock probes - ACTB, RAB7A =====Padlock probe for ACTB===== *'''ppACTB''' : /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATC CGACGG ACGTATCGGTAGTCGCAACGCA GGCAAAGGCGAGGCT --> 77bp **two arms : CTGTGCTCGCGGGGCG, GGCAAAGGCGAGGCT **site for decoding probes (dcProbe0-Cy3) : ACGTATCGGTAGTCGCAACGCA --> 22bp **site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp =====Padlock probe for RAB7A===== *'''ppRAB7A''' : /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATC CGACGG GTCTTGCGTGCGATACGGAGTA GAGGAGACTAAACGGAGGACA --> 90bp **two arms : GAAGCGAGAAGGTCCAAGTTCTG, GAGGAGACTAAACGGAGGACA **site for decoding probes (dcProbe1-Cy3) : GTCTTGCGTGCGATACGGAGTA --> 22bp **site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp =====2nd RCA probe===== *'''FISSEQ_ppRCA''' : GATATCGGGAAGCTGA*A*G --> 18bp ====Overall Process==== #Prepare each probes β the concentration of each probes are resuspended to 200uM for each ACTB, RAB7A and 2nd RCA primer. #Hybridize Padlock probe β mixture: 1uL ACTB + 1uL RAB7A + 98uL 2x SSC #Add mix, and incubate dish in the oven at 55C for 1hr. #Prepare Ampligase mix - 1uL AmpLigase + 10uL Buffer + 89uL H2O #Aspirate PBS in rolony sample and wash with PBS once #Add AmpLigase mix to sample dish, and incubate in the oven at 45C for 4hr. #Run 2nd RCA from Padlock probes #* Pre-anneal RCA primer (2uL of 100uM in 98uL 2x SSC) 60Β°C for 15min #* Add RCA master mix with aa-dUTP, run RCA for 20 hr. # Fix 2nd RCA with BS(PEG)9, and deactivate BS(PEG)9 with Tris pH8.0 treatment # Image ACTB on 2nd rolonies and Cy5 channel (1st rolonies) #* Decoding probe mixture: 10uL dcProbe0-Cy3 (10uM)+ 1uL Cy5-adapter for 1st rolony (100uM) + 89uL 2x SSC #* Pre heat probe mixture at 60C for 5min #* Add probe mixture to rolony sample and cool down at RT for 10min #* Wash with PBS twice and add 2mL PBS, and imaging # Image RAB7A on 2nd rolonies and Cy5 channel (1st rolonies) #* Strip probes by adding 80% Formamide in 2x SSC (pre-heat at 60C for 5min, and add and incubate at RT for 10min, and wash with PBS) #* Decoding probe mixture: 10uL dcProbe1-Cy3 (10uM)+ 1uL Cy5-adapter for 1st rolony (100uM) + 89uL 2x SSC #* Pre heat probe mixture at 60C for 5min #* Add probe mixture to rolony sample and cool down at RT for 10min #* Wash with PBS twice and add 2mL PBS, and imaging *2nd Rolony generation at 09/10~09/11, imaging at 09/11
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