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Abstract

Methods for preparing strand-specific sequencing libraries of oligonucleotides using an RNA polymerase promoter to re-transcribe antisense cDNA which has been reverse transcribed from mRNA are provided. The transcription step linearly amplifies sRNA prior to production of double-stranded cDNA to be sequenced and may be sufficient to eliminate the conventional PCR amplification step prior to sequencing. The methods incorporate anchor sequences, amplification sequences and other sequences required for a particular sequencing system or reaction by hybridization and extension of primers, and transcription of RNA, rather than ligation, thus reducing the number of steps and the time required for sample preparation for sequencing of RNA. Use of primer hybridization and transcription reactions in the methods also results in a library that exhibits reduced 3′ sequence bias.

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