What are the steps involved in RNA-Seq?

The RNA-seq protocol involves several key steps:

RNA Extraction: RNA is extracted from the sample and purified using RNA lysis buffer and organic solvent-based RNA isolation methods. 

RNA Fragmentation: The RNA is fragmented to prepare it for sequencing.

cDNA Synthesis: The fragmented RNA is converted into complementary DNA (cDNA). Taq DNA polymerases are used to amplify first-strand cDNAs. 

Library Preparation: cDNA library is made up of a collection of total cDNA synthesized for sequencing. cDNA is prepared into a sequencing library by adding platform-specific adapter sequences and amplifying the cDNA. This stage may also include the addition of barcodes for multiplexing. The exact procedure used is very specific to the platform used. 

Sequencing: The cDNAs in the cDNA library are sequenced using a next-generation sequencing (NGS) platform. This sequencing process is similar to the process used in DNA sequencing. 

RNA-seq Analysis: Sequencing reads may be aligned and compared to a reference genome for testing the presence of certain genes/RNA. Alternatively, sequence reads may be assembled for obtaining the complete sequence of the test RNAs. 

Various software tools are then used to quantify transcription levels and analyze alternatively spliced genes and other characteristics.