Preparation of cDNA libraries for high-throughput rna sequencing analysis of RNA 5′ ends

We provide a detailed protocol for preparing cDNA libraries suitable for high-throughput sequencing that are derived specifi cally from the 5′ ends of RNA (5′ specifi c RNA-seq). The protocol describes how cDNA libraries for 5′ specifi c RNA-seq can be tailored to analyze specifi c classes of RNAs based upon the phosphorylation status of the 5′ end. Thus, the analysis of cDNA libraries generated by these methods provides information regarding both the sequence and phosphorylation status of the 5′ ends of RNAs. 5′ specifi c RNA-seq can be used to analyze transcription initiation and posttranscriptional processing of RNAs with single base pair resolution on a genome-wide level.