title: Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts.
abstract: The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D014158:Transcription, Genetic / Q000235:genetics*