METHODS FOR PRODUCING STRANDED cDNA LIBRARIES

A method for selecting a particular strand of an RNA molecule in an RNA sample comprising:

• (a) hybridizing a first primer to an RNA sample under conditions wherein a complex is formed between a 3′ region of said first primer and an RNA molecule in said RNA sample, wherein said 3′ region of said first primer comprises a random nucleotide sequence and a first nucleotide sequence tag;
• (b) extending said primer of said complex by reverse transcription, thereby generating a complementary DNA (cDNA) molecule of said RNA molecule;
• (c) attaching a double stranded polynucleotide molecule to said cDNA molecule under conditions wherein the 5′ end of said double stranded polynucleotide molecule is attached to said cDNA molecule and said RNA molecule is not attached to the 3′ end of said double stranded polynucleotide molecule, and wherein said double stranded DNA molecule comprises a second nucleotide sequence tag;
• (d) removing said RNA molecule;
• and
• (e) selecting for the particular cDNA strand of the RNA molecule by hybridizing a probe or second primer to said first or second nucleotide sequence tags.

A method for creating a cDNA library representing a particular strand of a RNA molecule in an RNA sample comprising:

• (a) hybridizing a plurality of first primers to an RNA sample under conditions wherein complexes are formed between a 3′ region of two or more first primers in said plurality of first primers and two or more RNA molecules in said RNA sample, wherein said 3′ region of said first primers comprise a random nucleotide sequence and a first nucleotide sequence tag;
• (b) extending said plurality of first primers of said complexes by reverse transcription, thereby generating complementary DNA (cDNA) molecules of said two or more RNA molecules;
• (c) hybridizing a plurality of double stranded polynucleotide molecules comprising a second nucleotide sequence tag to said two or more cDNA molecules under conditions wherein:
• (i) a complex is formed between a 3′ overhang of a double stranded polynucleotide molecule in said plurality of double stranded polynucleotide molecules and a 3′ region of said cDNA molecule, wherein said 3′ overhang comprises a second random nucleotide sequence, and
• (ii) a 5′ end of a complementary second strand of said double stranded polynucleotide molecule in said plurality of double stranded polynucleotide molecules is adjacent to a 3′ end of said cDNA molecule;
• (d) attaching said 5′ end of said complementary second strand of said double stranded polynucleotide molecule to said 3′ end of said two or more cDNA molecules, thereby generating unattached strands of said double stranded polynucleotide molecules;
• (e) removing said unattached strands said double stranded polynucleotide molecules, thereby forming a plurality of single stranded cDNA molecules comprising a first and a second nucleotide sequence tag;
• and
• (f) converting said plurality of single stranded cDNA molecules to double stranded cDNA molecules, thereby creating a cDNA library representing a particular strand of a RNA molecule of in an RNA sample.

A method for creating a cDNA library representing a particular stand of a RNA molecule in an RNA sample comprising:

• (a) hybridizing a plurality of first primers to an RNA sample under conditions wherein complexes are formed between a 3′ region of two or more first primers in said plurality of first primers and two or more RNA molecules in said RNA sample, wherein said 3′ region of said single stranded primers comprise a random nucleotide sequence and a first nucleotide sequence tag;
• (b) extending said first primers of said complexes by reverse transcription, thereby generating complementary DNA (cDNA) molecules of said two or more RNA molecules;
• (c) attaching double stranded polynucleotide molecules to said cDNA molecules under conditions wherein the 5′ end of said double stranded polynucleotide molecules are attached to said cDNA molecules and said RNA molecules are not attached to the 3′ end of said double stranded polynucleotide molecules, wherein said double stranded DNA molecules comprise a second nucleotide sequence tag;
• (d) removing said RNA molecules;
• and
• (e) synthesizing complementary second strand DNA molecules from said cDNA molecules, thereby forming a cDNA library representing a particular strand of an RNA molecule in an RNA sample.