T7 mmachine kit

sgRNAs were designed targeting meis1 and pbx3 using CRISPRSCAN (https://www.crisprscan.org/?page=gene) (options: organism = ‘Frog-Xenopus tropicalis’, Gene = ‘pbx3’ or ‘meis1’, Cas9-NGG, In Vitro T7 promoter). PCR was performed as described in Bhattacharya et al. (2015) (link). SgRNA was transcribed using T7 mMachine kit (Ambion). Guides were injected into 2 blastomeres of 4 cell embryos with 1.5 ng Cas9 (Bhattacharya et al., 2015 (link); Nakayama et al., 2013 (link)). All guides were injected at a dose of 400 pg/embryo.

The UT-A3 (AF258602), UT-A2 (AF367359) and UT-B (AF448798) were a gift from Dr Bryce MacIver, Harvard Medical School, MA, USA. DNA sequences encoding C-terminally c-Myc tagged murine UTs: mUT-B, mUT-A2 and mUT-A3 were subcloned into the P7TS expression vector. The resulting plasmids were transformed into TOP10 competent cells via heat shock, and purified with a DNA Miniprep kit (part #28104, Qiagen, Valencia, CA, USA). All of the plasmids were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (part #4337455, Applied Biosystems, Foster City, CA, USA) and an ABI Prism 3130XL Genetic Analyzer (HITACHI, Tokyo, Japan).
All UT-encoding cDNAs were linearised with XbaI restriction enzyme (part #R0145S, New England Biolabs, Ipswich, MA, USA) and purified using the QIAquick PCR Purification Kit (part #27106, Qiagen). The linearised and purified DNAs were transcribed into capped RNA (cRNA) using the T7 mMachine Kit (part #AM1344, Ambion, Austin, TX, USA) and the cRNAs were purified with the RNeasy MinElute RNA Cleanup Kit (part #74204, Qiagen). The concentration and purity of all DNAs and RNAs were quantified using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).