RNA sequencing (RNA-Seq) is a powerful technique to study the transcriptome of an organism at a given moment. As its name suggests, RNA-Seq is sequencing the RNA molecules from the sample. But how are the samples prepared? Here I will present a summary of this process:

Disclaimer: This post is not a guide or protocol to perform RNA extraction for RNA-Seq. The objective giving an overview of the process, highlighting the most important steps. 

1. Sample homogenization: The first step consists of “breaking” the cells to release the nucleic acids. This step can be done using liquid nitrogen, a mortar, and a pestle and then diluting the grounded sample in a buffer. If you then centrifuge the mix, the nucleic acids will be in the supernatant.
2. RNA extraction: To separate the RNA from other sample components exist different commercial kits. Most of them use silica columns that bind nucleic acids. To separate RNA from DNA they use specific columns that bind only DNA.
3. DNA elimination: It is necessary removing all traces of DNA (otherwise, you might end up sequencing DNA instead of RNA!). The most effective approach is treating the sample with DNase, an enzyme that diggest only DNA.
4. rRNA depletion: A considerable amount of the total RNA in the cells correspond to ribosomic RNA (rRNA). This type of RNA, together with some proteins, form the ribosomes. Sequencing these RNAs does not provide any information on the expression of the different genes, and therefore they should be depleted. For this purpose, there are commercial kits. Another approach is using probes complementary in sequence to the rRNA with biotin. These probes would join the rRNA molecules due to the complementarity. Then they would be removed using streptavidin (streptavidin binds biotin).
5. Fragmentation: Most of the current DNA sequencing methods cannot directly sequence only relatively short (300-1000 nucleotides long) DNA fragments in a single reaction. Therefore, it is necessary to diggest the RNA molecules into smaller fragments. For this purpose, you can use an RNase (an enzyme that cuts RNA).
6. cDNA library preparation: At this step, the set of (single strand) RNA fragments in the sample serve as a template to generate (double strand) DNA fragments. This DNA is the complementary DNA (cDNA). The enzyme catalysing this reaction is the reverse transcriptase (RT).
7. Sequencing: The cDNA library is sequenced using DNA sequencing methods. The result you obtain is a list of all the DNA sequences in your library.
8. Data analysis: Finally, you map the reads from the sequencing against the genome of the organism you are studying. Genes with a high number of matching sequences are more expressed in the studied sample.

Final remark: During the whole process, you need to be extremely careful not to contaminate the samples or their degradation by RNases. These enzymes degrade the RNA and are literally everywhere. Keeping your material clean and your samples in ice (the optimal temperature for the RNAse reactions is around 37ºC) is essential.