Methods of small-RNA transcriptome sequencing and applications thereof

A method of treating, detecting, or preparing an RNA sample comprising the steps of:

• (a) providing a sample containing one or more ribosomal RNA molecules and one or more small RNA molecules;
• (b) masking said ribosomal RNA molecules by providing one or more oligonucleotide(s) complementary to said ribosomal RNA molecules;
• (c) contacting two or more 3′ oligonucleotide adaptors with a 3′ end of said small RNA molecules, wherein the 5′ end of the two or more 3′ oligonucleotide adaptors is adenylated and wherein the 3′ end of the two or more 3′ oligonucleotide adaptors is blocked by a first chemical group, to obtain a corresponding ligated 3′ oligonucleotide adaptor linked to said small RNA molecules and a corresponding unreacted free form 3′ oligonucleotide adaptor;
• (d) hybridizing two or more RT primer oligonucleotides to said 3′ ligated oligonucleotide adaptor and said free form 3′ oligonucleotide adaptor, wherein the 5′ end of the RT primer is blocked by a second chemical group, and wherein said free form 3′ oligonucleotide adaptor is converted to a dimer of said free form 3′ oligonucleotide and said RT primer oligonucleotide;
• (e) deadenylating the 5′ end of the two or more 3′ oligonucleotide adaptors;
• (f) removing said dimer by digest with lambda exonuclease;
• (g) ligating a 5′ oligonucleotide adaptor to a 5′ end of said small RNA molecules;
• (h) performing reverse transcription of said small RNA molecules;
• and
• (i) performing a first PCR reaction with a RT primer and a RP1 primer and a second PCR with RP1 and indexed primers.