We will provide an overview of the experimental and computational steps involved in RNA-seq for both bulk and single-cell experiments. We will begin with a brief review of Illumina short-read sequencing by synthesis; continue to describing the molecular biology used in preparing RNA-seq libraries; and discuss quality trimming, read alignment, transcript quantification and normalization of gene expression measures. We will conclude with a discussion of techniques commonly leveraged in single-cell RNA-Seq: linear pre-amplification, unique molecular identifiers (UMI/RMTs) and 3’-barcode counting. Throughout the primer, we will mention potential sources of bias that can be introduced at each step and why they occur.
MIA Talks
Experimental and computational techniques underlying RNA-seq
September 28, 2016
Dept. of Systems Biology, Harvard University; Harvard-MIT Health Sciences and Technology