Fine Expression Profiling of Full-length Transcripts using a Size-unbiased cDNA Library Prepared with the Vector-capping Method

Abstract

Recently, we have developed a vector-capping method for constructing a full-length cDNA library. In the present study, we performed in-depth analysis of the vector-capped cDNA library prepared from a single type of cell. As a result of single-pass sequencing analysis of 24 000 clones randomly isolated from the unamplified library, we identified 19 951 full-length cDNA clones whose intactness was confirmed by the presence of an additional G at their 5' end. The full-length cDNA content was >95%. Mapping these sequences to the human genome, we identified 4513 transcriptional units that include 36 antisense transcripts against known genes. Comparison of the frequencies of abundant clones showed that the expression profiles of different libraries, including the distribution of transcriptional start sites (TSSs), were reproducible. The analysis of long-sized cDNAs showed that this library contained many cDNAs with a long-sized insert up to 11 199 bp of golgin B, including multiple slicing variants for filamin A and filamin B. These results suggest that the size-unbiased full-length cDNA library constructed using the vector-capping method will be an ideal resource for fine expression profiling of transcriptional variants with alternative TSSs and alternative splicing.

Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto

“…Full-length cDNA clones of medaka cpsf6 provided from NBRP Medaka [ 36 , 39 ], oleb23l11 and oleb36o05 with 587b and 2004b 3'UTR respectively, were sequenced (GenBank LC147085, LC148409) and 10 exons with 1656bp Open Reading Frames (ORF) spanning exon 1~9 were determined. Two genomic sequence sets corresponding to the exon 1~9, including splicing acceptor and donor sites, were amplified from the genomic DNA of 10 embryos taken from nar mutants and also 10 embryos of the Cab wild type, using Ex-Taq polymerase (Takara, Kyoto, Japan) and the DNA sequences of the PCR amplified fragments were determined according to Takehana et al (2005) [ 40 ].…”

“…Capped RNAs were synthesized from the above mentioned full-length medaka cpsf6 cDNA clones ( oleb23l11 and oleb36o05 ). The cDNA clones have artificial poly A sequences in the downstream of the 3'UTR [ 39 ]. The cDNA clones were linearized at NotI sites located just downstream of the poly A sequences, and were used as a template for in vitro transcription by T7 RNA polymerase using an mMessage mMachine kit (Ambion, Texas, USA).…”