RNa-seq Protocol


Taken from Stark et al. “RNA sequencing: the teenage years.” Nat Rev Genet. 2019 20(11):631-656. “RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs.” With considerable technical progress and constant decreasing costs, RNA-Seq has became a standard technique in biology to study gene expression. This protocol describes how total RNA are converted to short sequences called “reads” that can in turn be used to get insights into gene expression. Through careful experimental design, these gene expression information can yield new research avenues and answer crucial questions. This protocol does not deal with the wet-lab section of RNA-seq i.e. total RNA isolation or library preparation but rather explores the dry-lab parts of RNA-seq that include bioinformatics . A regularly updated tutorial is available in the form of a Carpentry lesson website: https://scienceparkstudygroup.github.io/rna-seq-lesson/index.html

RNA Sequencing techniques

This short video gives you a short introduction to the techniques of RNA sequencing:

  • Assessing the quality of the input RNA,
  • Protocols to prepare a sequencing RNA library (Illumina example),
  • Illumina Next Generation “sequencing by synthesis” workflow,
  • Output of the sequencing process, quality check of the produced sequences.
After watching this video, please refer to these RNA-seq lesson sections:

differential gene expression analysis

This video addresses the main steps performed after the bioinformatic parts of RNA-seq. It shows the main inputs (gene counts, sample to conditions) and the main outputs (table of differential genes, volcano plot) that one can get from a classical differential expression analysis.

Please refer to the RNA-seq lesson section “Differential expression” for additional info and practical know-how.