1. Site Protocol component 1234567891011121314151617181920212223 NMD inhibitor Cyclo- heximide 100  g/ml 4hrs no cell culture Cyclo- heximide 100  g/ml 4hrs Puromycin 200  g/ml 4-6 hours Cyclo- heximide (250  g/ml l) 4 hrs Cyclo- heximide 100  g/ml 4hrs Cyclo- heximide 100  g/ml 4hrs Puromycin 100μg/ml 8 hours Puromycin 200μg/ml 4 hours no cell culture Puromycin 250  g/ml or 4- 6 hours Cyclo- heximide 100  g/ml 4hrs Puromycin 10  g/ml no cell culture no cell culture Cyclo- heximide 100  g/ml 4hrs Cyclo- heximide 100μg/ml 4 hours None Cyclo- heximide 100  g/ml 4 hours None Cyclo- heximide 100  g/ml 4 hours Cyclo- heximide 200  g/ml 4 hours Cyclo- heximide 100  g/ml 4 hours RNA extraction Rneasy mini kit MagNa Pure TrizolTripureTrizol Rneasy mini kit Rneasy mini kit Trizol Rneasy mini kit Trizol Rneasy mini kit Trizol Rneasy mini kit RNAII, MN Rneasy mini kit RNAqueous Rneasy mini kit Rneasy mini kit Rneasy mini kit Rneasy mini kit DNAse treatment rDnaseDNase1No RNeasy Minikit, RNase-Free DNase NoDNase1 RNeasy Minikit, RNase-Free DNase No RNeasy Minikit, RNase-Free DNase NoDNAse1No RNeasy Minikit, RNase-Free DNase RNAII, MN (include DNase treatment) Turbo DNA- free RNAqueous DNAseI NorDnase Minikit, RNase-Free DNase ABI cDNA synthesis primer Random hexamers Random hexamers / Oligo d(T) Anchored Oligo d(T) Random hexamers Random hexamers / Oligo d(T) Oligo d(T) Random hexamers Oligo d(T) Random hexamers Gene specific Gene specific NKOligo d(T) Random hexamers cDNA synthesis kit Super- scriptIII PrimeScript RT reagent kit High Fidelity trantscriptior kit Superscript Firststrand Short: transcripts: High Capacity RNA-to- cDNA. Long: High fidelity transcriptor Super- scriptIII Super- scriptIII Super- scriptIII Super- ScriptIII Invitrogen SuperscriptII Super- scriptIII MMLV- Reverse transcriptase Super- scriptIII RevertAid Premium First Strand Super- scriptIII Super- scriptIII Super- scriptIII Super- scriptIII MMLV- Reverse transcriptase Super- scriptIII DNA polymerase Amplitaq Gold Hot Star Taq Platinum Taq DNA Polymerase Invitrogen Taq polymerase Amplitaq Gold Short: Ecotaq DNA pol Long: Expand Long Range Amplitaq Gold GoTaq Flexi Sigma taq polymerase Amplitaq Gold MegaMIX Double Amplitaq Gold Thermoprime FastStart Taq DNA Polymerase Phusion hot start Qiagen Taq Amplitaq Gold Platinum Taq Platinum Taq NKno Amplitaq Gold Amplitaq Gold (25 cycles) Detection method Agarose gel Capillary Gel EP Agarose gel PAA-gel silver staining Agarose gel Phase 1: Agarose gel and Agilent Bioanalyser Phase 2: Cap gel EP Qiaxel agarose gel BioanalyserAgarose gel MultiNAAgarose gel Labchip GX (capillary system) and agarose gel Agarose and Cap EP Analysis of PCR products Band excision, cloning, sequencing Capillary Gel EP, sequencing Band excision and sequencing Sequence PCR products directly Band excision, cloning, sequencing Sequence PCR products directly Sequence PCR products directly Agarose Gel EP, sequencing Qiaxel size visualisation Agarose gel & sequencing, Band excision and sequencing Band excision and sequencing PCR products directly Band excision and sequencing Band excision, cloning, sequencing Band excision and sequencing or sequenced PCR products directly Band excision and sequencing NK Band excision and sequencing Band excision and sequencing PCR products directly Direct sequencing Sub-cloning pGemTNo TopoTA cloning No TopoTA cloning pGemTNo pGemTNo Supplemental Data Table 1. Comparison of splicing assay protocols. As submitted by the 23 participating sites.