Illumina HiSeq 2500 sequencing; GSM1906977: Liver Nuc1; Mus musculus; RNA-Seq

Liver Nuc1

Messenger RNA is thought to predominantly reside in the cytoplasm, where it is translated and eventually degraded. Although nuclear retention of mRNA has a regulatory potential it is considered extremely rare in mammals. Here to explore the extent of mRNA retention in metabolic tissues we combine deep sequencing of nuclear and cytoplasmic RNA fractions with single molecule transcript imaging in mouse beta cells, liver and gut. We identify a wide range of protein coding genes for which the levels of spliced polyadenylated mRNA are higher in the nucleus than in the cytoplasm. These include genes such as the transcription factor ChREBP, Nlrp6, Glucokinase and Glucagon receptor. We demonstrate that nuclear retention of mRNA can efficiently buffer cytoplasmic transcript levels from noise that emanates from transcriptional bursts. Our study challenges the view that transcripts predominantly reside in the cytoplasm and reveals a role of the nucleus in dampening gene expression noise. Overall design: we have total of 8 samples all are mice. liver nuclear RNA (2 replicates), liver cytoplasmic RNA (2 replicates), MIN6 (cell line) nuclear RNA (2 replicates), MIN6 (cell line) cytoplasmic RNA (2 replicates)

Nuclear retention of mRNA in mammalian tissues

Submitted by Gene Expression Omnibus on 14-NOV-2015

Isolation of nuclear and cytoplasmic liver mRNA was performed according to the Nascent-SEQ protocol (Menet et al., 2012) except for minor modifications. In order to isolate cytoplasmic mRNA the supernatant was collected following nuclei isolation by sucrose gradient. For isolation of nuclear mRNA the supernatant was collected following chromatin isolation. 1/50 volumes of 5M Nacl and 2.5 volumes of 100% EtOH were added to the supernatants collected, and the mixture was incubated at -200C for 1 hour and then centrifuged for 20 minutes at full speed. The pellet was resuspended in 0.5 ml 0.5% SDS buffer (0.5% SDS, 0.1M NaCl, 1mM EDTA, 10mM Tris-HCl). Similar volume of acid phenol:chloroform (Ambion AM9722) mixture was added. The mixture was then vortexed and centrifuged at full speed for 5 minutes at RT. The aqueous phase was transferred to a new tube and 1 ml of 0.5% SDS buffer was added to the phenol phase for re-extraction. The two aqueous phases were combined and re-extracted with acid phenol:chloroform. The RNA from the aqueous phase was then isolated using standard EtOH precipitation. For isolation of nuclear and cytoplasmic RNA from MIN6, the cells (~2x106) were first trypsinized and washed with cold PBS. Cell pellet was then treated with 175 µl RLN buffer (Tris pH8.0 50mM, NaCl 140mM, MgCl2 1.5mM, NP-40 0.15mM, EDTA 10mM, DTT 1mM, RNase inhibitor 10U/ml) and incubated for 5 minutes on ice. Lysate was centrifuged at 300g for 5 minutes in a cold centrifuge. The supernatant (cytoplasmic fraction) was separated and the pellet was resuspended with same volume of RLN buffer and immediately centrifuged at 500g for 1 minute. Pellet was resuspended in 1 ml S1 buffer (sucrose 250mM, MgCl2 10mM, RNase inhibitor 10U/ml), layered over 3 ml of S3 buffer (sucrose 880mM, MgCl2 10mM, RNase inhibitor 10U/ml) and centrifuged for 10 minutes in cold centrifuge at 2800g. RNA from the pellet (nuclear fraction) and the cytoplasmic fraction was isolated using RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. Libraries were prepared with Illumina TrueSeq kits and sequenced on Illumina HiSeq.

Nuclear retention of mRNA in mammalian tissues

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