CAGE-1
http://metadb.riken.jp/db/SciNetS_ria187i/cria187s6ria187u30i
"The CAGE (cap analysis gene expression) tags are obtained by sequencing concatemers of DNA tags deriving from the initial 20/21 nucleotides from 5' end mRNAs, prepared in accordance to Shiraki et al. (Proc Natl Acad Sci U S A. 100, 15776-81, 2003).
At first step, full-length cDNAs were selected with the Cap-Trapper. Next, a specific linker (Linker1, which contains the ClassIIs restriction enzyme site MmeI) was ligated to the cDNA. Linker1 may contain extra 5 bp sequences tag, and 15 of such different sequences tags were used to tag different starting RNA samples. Then the second strand of cDNA synthesized. Resulting double-stranded cDNAs were cleaved by the restriction enzyme MmeI and a second linker (Linker2) was ligated to the 2 bp overhang at the MmeI cleaved site, to produce a 5' 20/21 tag having two linkers at both sides. The ligation products were separated from unmodified DNA with magnetic beads. The 5' end cDNA tags were released from the beads, and the DNA fragments were amplified in a PCR step by using the two linker-specific primers (Primer1 (uni-PCR), Primer2 (MmeI-PCR)). The desired 32-37 bp tags were purified and ligated to form concatemers, and then the concatemer were fractionated and ligated to the plasmid ZErO-1. The ligations were finally electroporated into DH10b cells (Invitrogen) and obtained plasmids were sequenced with forward primers essentially as described with minor modifications to use zeocin for selection of recombinants. Each CAGE tag comprises short sequences of about 20 bp derived from the 5' end of a full-length cDNA. The length of a CAGE tag may vary due to the limited specificity of MmeI. Note that up to 70% of the CAGE tags may have an unspecific G in the first position at the 5' end.
Linker1: ""Upper oligonucleotide GN6"": biotin-agagagagacctcgagtaactataacggtcctaaggtagcgacctagg (5 bp) tccgacGNNNNN and ""Upper oligonucleotide N6"": biotin-agagagagacctcgagtaactataacggtcctaaggtagcgacctagg (5 bp) tccgacNNNNNN were mixed. ""Lower oligonucleotide"": phosphate group-gtcgga (5 bp) cctaggtcgctaccttaggaccgttatagttactcgaggtctctctct-NH2
Linker2: ""Upper-XmaJI"": Pi-cctaggtcaggactcttctatagtgtcacctaaagacacacacac -NH2 ""Lower-XmaJI"": gtgtgtgtgtctttaggtgacactatagaagagtcctgacctaggNN
Primer1 (uni-PCR): 5'-biotin-GTGTGTGTGTCTTTAGGTGACACTA-3'
Primer2 (MmeI-PCR): 5'-biotin-AGAGAGAGACCTCGAGTAACTATAA-3'"