The immune system operates at the scale of the whole organism in mammals. We currently lack experimental approaches to systematically track and study organism-wide molecular processes in mice. Here we describe an integrated toolkit for measuring gene expression in whole tissues, 3-prime mRNA extension sequencing, that is applicable to most mouse organs and any mouse model of interest. Further, the methods of RNA-seq described in this protocol are broadly applicable to other sample types beyond whole organs, such as tissue samples or isolated cell populations. We report procedures to collect, store and lyse a dozen organ types using conditions compatible with the extraction of high-quality RNA. In addition, we detail protocols to perform high-throughput and low-cost RNA extraction and sequencing, as well as downstream data analysis. The protocol takes 5 d to process 384 mouse organs from collecting tissues to obtaining raw sequencing data, with additional time required for data analysis and mining. The protocol is accessible to individuals with basic skills in (i) mouse perfusion and dissection for sample collection and (ii) computation using Unix and R for data analysis. Overall, the methods presented here fill a gap in our toolbox for studying organism-wide processes in immunology and physiology.
Overall design: To study immune processes at the organism-level, four replicates of 6 weeks old female mice were anasthesized, and transcardially perfused with PBS supplemented with 1 mM EDTA. 15 tissues (Liver, bone marrow, lung, inguinal lymph node, mesenteric lymph node, colon, small intestine, female reproductive tract, brain, kidney, thymus, peripheral blood mononuclear cells, heart, skin, spleen) were collected and stored in RNAlater at 4C overnight before freezing at -80C for long-term storage.
Less...