Construction of the libraries is described in Kitayama et al. (in preparation). They were arrayed in NIBB and sequenced by NIG.

Briefly, cDNA libraries were constructed with ZAP-cDNA Gigapack III cloning kit (Stratagene) according to manufacturer's instruction with minor modification. 5 ug of poly A+ RNA from each of the three embryonic stages was oligo dT primed (XhoI dT primer). After second strand synthesis, EcoRI adapters were ligated and the cDNA was directionally cloned with EcoRI at the 5' end and XhoI at the 3' end. Normalization was carried out with ssDNA phagemids hybridized to biotinylated driver, which was prepared from the same library by PCR. The average insert size of about 1.5kb. The vector used is pBlueScript SK(-) (Stratagene) and the host bacteria is DH10B (GIBCO).

Primers for PCR amplification:
T3 primer:        ATT AAC CCT CAC TAA AG (17mer)
T7 primer:        AAT ACG ACT CAC TAT AG (17mer)

XL001~XL050; NIBB Mochii-normalized Xenopus neurula library
XL051~XL110; NIBB Mochii-normalized Xenopus tailbud library
XL111~XL220; NIBB Mochii-normalized Xenopus early gastrula library

Construction of the library is described in Hyodo-Miura et al. (in preparation ). They were arrayed in NIBB and sequenced by NIG as the National Bio Resource Project.

About 1000pieces of Keller explants were dissected at stage 10.5 and cultured in 0.1% BSA/1 x steinberg's solution. Total RNA was isolated by the acid guanidinium thiocyanate-phenolchloroform method from explants at stage 11~15. A cDNA library was made with the ZAP-cDNA Synthesis Kit (Stratagene ) according to manufacturer's instruction with minor modification. 4ug of polyA+RNA was oligo dT primed (XhoI dT primer). After second strand synthesis, EcoRI adaptor was ligated and the cDNA was directionally cloned with EcoRI at the 5' end and XhoI at the 3' end into the pCS2p+ vector. The average insert size was about 2kb. The host bacteria was XL2-Blue (Stratagene ).

XL221 ~ XL341; Yamamoto / Hyodo-Miura NIBB/NBRP Xenopus DMZ library

Construction of the libraries is described in Osada et al. (Development 130, 1783-1794, 2003). They were arrayed in NIBB and sequenced by NIG as the National Bio Resource Project.

About 500 pieces of anterior neural plates were dissected from stage 12-12.5 embryos. The neuroectoderm layer was separated from the underlying mesendoderm layer in 1 x modified Barth's solution containing 1-2 mg/ml collagenase. After poly (A)+RNA selection by an oligo(dT) cellulose column, a cDNA library was made with the SuperScript plasmid system for cDNA synthesis and plasmid cloning (Invitrogen) and the pCS105 vector (Hsu D.R. et al., Mol Cell. 1, 673-83, 1998). The first strand cDNA was synthesized with oligo dT-NotI primer. After second strand synthesis, SalI adapter (gtcgacCCACGCGTCCG; the lower case letters show SalI site) were ligated and the cDNA was directionally cloned with SalI at the 5' end and NotI at the 3' end. The average insert size is about 2kb and the host bacteria is XL1 blue (Stratagene) (?).

XL401~XL520; Osada/Taira NBRP Xenopus ANE library