To determine the complete structure of the retrocopy-associated transcripts, we performed 5′ and 3′ RACE using a SMARTer RACE 5′/3′ Kit (Clontech) according to the manufacturer’s instructions. The gene specific primer sequences and the derivation of the RNAs that were used for RACE experiments are listed in supplementary table S3 , Supplementary Material online. The PCR products were cloned using a pTAC-2 vector (BioDynamics Laboratory) and ECOS-competent Escherichia coli DH5α (NIPPON GENE). Plasmids were purified using FastGene Plasmid Mini (NIPPON Genetics) and sequenced.
Ecos competent escherichia coli dh5α
Manufactured by Nippon Gene
ECOS-competent Escherichia coli DH5α is a strain of E. coli that has been genetically modified to be highly competent for transformation. It is commonly used in molecular biology research and applications.
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3 protocols using ecos competent escherichia coli dh5α
1
Characterization of Retrocopy-Associated Transcripts
2
Bisulfite Sequencing of Genomic DNA
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Genomic DNA was extracted from yolk sac placentas using Trizol (Life Technologies). Genomic DNA was treated with sodium bisulphite solution, as described previously [45 (link), 46 (link)]. After the bisulphite treatment of the genomic DNA, 35 cycles of PCR were carried out using the primers designed by MethPrimer [47 (link)]. The PCR products were cloned using a pTAC-2 vector (BioDynamics Laboratory) and ECOS-competent Escherichia coli DH5α (NIPPON GENE). Plasmids were purified using FastGene Plasmid Mini (NIPPON Genetics) and sequenced. The sequence data were analysed by QUMA (quantification tool for methylation analysis; http//quma.cdb.riken.jp) [48 (link)]. Primer sequences used in this study are provided in the Additional file 1 .
3
Determining ALID Structure via RACE
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To determine the complete structure of ALID, we performed 5′ and 3′ RACE experiments using a SMARTer RACE 5′/3′ Kit (Clontech) according to the manufacturer’s instructions. The RACE products amplified from the templates created using pouch young brain RNA were cloned using a pTAC-2 vector (BioDynamics Laboratory) and ECOS-competent Escherichia coli DH5α (NIPPON GENE). Plasmids were purified using FastGene Plasmid Mini (NIPPON Genetics) and sequenced. Primer sequences used in this study are provided in the Additional file 1 .
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