BioSample

10 ug of polyA+ RNA was isolated from a mixture of embryos at stage 10, 20 and 30 and primed by oligo-dT primer: 5'-GAGAGAGAGAAGGATCC(T)16VN-3' (where V=G,A,C). 5-methyl-dCTP was used instead of dCTP in the first-strand synthesis in order to get hemimethylated cDNA. After full-length enrichment, oligo-dG tailing and normalization against itself, second-strand synthesis was carried out by priming with 5'-GAGAGAGAGACTCGAGTTAATTAAT(C)13-3'. dsDNA was digested with XhoI/BamHI and directionally cloned into the pRKW2 vector. Average insert size is 1.5 kb. Library constructed using the Carninci protocol (Genome Research 2000) by Drs. W. Wu and C. Niehrs (DKFZ, Heidelberg, Germany).