cDNA Sequencing To generate cDNA, RNA was isolated from frozen tissue with the RNeasy minikit (Qiagen), treated with DNase I to degrade residual DNA and complementary DNA was reverse transcribed in vitro using the High Capacity cDNA Reverse Transcription Kit

cDNA Sequencing To generate cDNA, RNA was isolated from frozen tissue with the RNeasy minikit (Qiagen), treated with DNase I to degrade residual DNA and complementary DNA was reverse transcribed in vitro using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Where required, normal and polyp tissue was microdissected with the laser capture PALM system. DNA was extracted using the PicoPure DNA extraction kit (Arcturus). Sequencing of cDNA/gDNA was carried out using the 2x Big Dye Terminator v3.1 reagent (Applied Biosystems). Unincorporated dye terminators were removed with the DyeEx 2.0 Spin kit (Qiagen) and the purified products were run on the ABI 3730 DNA analyser (Applied Biosystems). Primers used to screen the hotspot mutation regions in Apc, Tp53, Ctnnb1 and Kras are available on request. PCR products from Ctnnb1 mutant polyps were TA cloned (Promega) and DNA extracted from individual colonies (Lyse and Go, Thermo Scientific) which were subsequently sequenced.