Abstract
We provide a detailed protocol for preparing cDNA libraries suitable for high-throughput sequencing that are derived specifically from the 5' ends of RNA (5' specific RNA-seq). The protocol describes how cDNA libraries for 5' specific RNA-seq can be tailored to analyze specific 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' specific RNA-seq can be used to analyze transcription initiation and posttranscriptional processing of RNAs with single base pair resolution on a genome-wide level.