Expression profiling by high throughput sequencing
Summary
Cooling patients to sub-physiological temperatures is an integral part of modern medicine. We show that cold exposure induces temperature-specific changes to the higher-order chromatin and gene expression profiles of human cells. These changes are particularly dramatic at 18°C, a temperature synonymous with that experienced by patients undergoing controlled deep-hypothermia during surgery. Cells exposed to 18°C exhibit largely nuclear-restricted transcriptome changes. These include the nuclear accumulation of transcripts of genes of the negative limbs of the core circadian clock, most notably REV-ERBα. This response is accompanied by compaction of higher-order chromatin and hindrance of mRNPs from engaging nuclear pores. Rewarming reverses chromatin compaction and releases the transcripts into the cytoplasm, triggering a pulse of negative limb gene proteins that resets the circadian clock. We show that cold-induced upregulation of REV-ERBα is sufficient to trigger this resetting. Our findings uncover principles of the cellular cold-response that must be considered for current and future applications involving therapeutic deep-hypothermia.
Overall design
RNA was extracted from nuclear and cytoplasmic subcellular fractions after exposing two different cell lines (AC16 and U2OS cells) to different temperature conditions. For Drosophila melanogaster spiked-in samples, RNA was also extracted from nuclear and cytoplasmic fractions of AC16 cells exposed to different temperature conditions spiked-in with a known ratio of Drosophila melanogaster Schneider 2 cells prior to fractionation. The 3’ ends of extracted RNA were generated into libraries using the QuantSeq 3’mRNA-Seq library kit for Ion Torrent (Lexogen). Libraries for full-length RNA-seq were prepared using the Ion Total RNA-seq Kit v2 (ThermoFisher). For this full-length RNA-seq polyadenylated RNAs were first purified from total RNA from each fraction using the NEBNext Poly(A) mRNA Isolation Module. Libraries were loaded onto the Ion Chef System (ThermoFisher) for template preparation and chip loading and the resulting chips were sequenced on the Ion Proton Sequencing System (ThermoFisher). Sequences were aligned using the Ion Torrent Server TMAP aligner to genome build hg19 or to a combined hg19 dm6 genome build for Drosophila melanogaster spiked in samples.
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