With the rapid development of the next-generation sequencing (NGS) technology, high-throughput RNA sequencing or RNA-seq is becoming a key experimental approach in the study of gene expression and transcriptome. The overwhelming amount of RNA-seq data brings new opportunities and challenges for bioinformatics. The efficient and effective processing and analysis of RNA-seq data is becoming the bottleneck for turning the possibilities provided by the new technology into real scientific discovery. A general description of the typical RNA-seq protocol was given. A complete review of major methods and available software in the processing and analysis of RNA-seq data were presented, using the Illumina/Solexa platform as an example. Questions that are still open and awaiting further research are also discussed.