Mmessage mmachine t7 transcription kit

LncRNA knockout models were produced in the Transgenic Unit of the Institute of Molecular Genetics ASCR, Czech Centre for Phenogenomics using Cas9-mediated deletion of lncRNA promoters (Supplementary Figs. S7 and S8).¹⁵ (link)^,16 All animal experiments were approved by the Institutional Animal Use and Care Committees (project number 58-2015) and were carried out in accordance with the law.
Sequences of guide RNAs are listed in the Table S5. To produce guide RNAs, synthetic 128 nt guide RNA templates including T7 promoter, 18 nt sgRNA and tracrRNA sequences were amplified using T7 and TracrRNA primers (Supplementary Table S5). Guide RNAs were produced in vitro using the Ambion mMESSAGE mMACHINE T7 Transcription Kit, and purified using the mirPremier microRNA Isolation Kit (Sigma). The Cas9 mRNA was synthesized from pSp Cas9-puro plasmid using Ambion mMESSAGE mMACHINE T7 Transcription Kit, and purified using the Qiagen RNasy mini kit. A sample for microinjection was prepared by mixing two guide RNAs in ultra-pure water at a concentration of 25 ng/µl for each one together with Cas9 RNA (100 ng/µl) . Five picoliters of the microinjection mixture were injected into male pronuclei of C57Bl/6 zygotes and transferred into pseudopregnant recipient mice. PCR genotyping was performed on tail biopsies from 4 weeks-old animals. Primers are listed in Supplementary Table S5.

The Sirena1 deletion mutant model was produced in the Czech Centre for Phenogenomics at the Institute of Molecular Genetics ASCR using Cas9-mediated deletion of the Sirena1 promoter (13 (link)). Sequences of guide RNAs are listed in Supplementary Table S1. To produce guide RNAs, synthetic 128 nt guide RNA templates including T7 promoter, 18nt sgRNA and tracrRNA sequences were amplified using T7 and tracrRNA primers. Guide RNAs were produced in vitro using the Ambion mMESSAGE mMACHINE T7 Transcription Kit, and purified using the mirPremier™ microRNA Isolation Kit (Sigma). The Cas9 mRNA was synthesized from pSpCas9-puro plasmid using the Ambion mMESSAGE mMACHINE T7 Transcription Kit, and purified using the RNeasy Mini kit (Qiagen). A sample for microinjection was prepared by mixing two guide RNAs in water (25 ng/μl for each) together with Cas9 mRNA (100 ng/μl). Five picoliters of the mixture were microinjected into male pronuclei of C57Bl/6J zygotes and transferred into pseudo-pregnant recipient mice. PCR genotyping was performed with tail biopsies from four-week-old animals (primers are listed in Supplementary Table S1). We obtained seven positive founders, of which one transmitted the mutant allele to F₁. After two generations of breeding with C57Bl/6NCrl animals, the heterozygotes were used for breeding Sirena1^−/− animals for phenotype analysis.