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===Day 2=== #Wash with 1X PBS twice #Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT #Wash with 1X PBS twice #Quench with 1M Tris for 30min at RT #Wash with 1X PBS twice #RNA Removal #*Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C #Wash with H2O twice #Add 200ul CircLigaseII mix and incuabate 3hr at 60C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||640 |- | CircLigase Buffer 10X||100 |- | MnCl2 50mM||50 |- | Betaine 5M|| 200 |- | CircLigase II (100U/ul)||10 |- | Total||1000 |} #Wash with 1X PBS twice #Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C #Wash with 2X SSC + 30% formamide for 10min at 60C #wash with 2X SSC, 1X SSC, 1X PBS once each #Add 200ul RCA mix and incubate for 15hr at 30C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||174 |- | 10X Phi29 Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | Phi 29 (low conc)||2 |- | Total||200 |}
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