title: GSM5538519: S25: Unf_Total_ZT00_S1_WT; Mus musculus; RNA-Seq
library_construction_protocol: Pieces of the big lobe of the mouse liver were dissected. RNA from unfractionnated liver: pieces of liver were snap-frozen in liquid nitrogen and extracted on spin-columns from miRNEasy kit  following manufacturer's instruction (QIAGEN, Germany). Nuclear and cytoplasmic RNA were extracted following the protocole of nascent-seq described in Menet et al. 2012. Briefly, liver pieces were homogenised in a 2.2M sucrose buffer, layered on top of 2.05M sucrose cushion and ultra-centrifuged. Cytoplasmic RNA in sucrose was precipitated in ethanol 70% overnight at -80° and extracted on spin-columns from the miRNEAsy kit (Qiagen). Nuclear RNA in the pellet was resuspended in a guanidium thiocyanate-containing extraction buffer and collected on spin-column from the miRNEasy kit (Qiagen). Additional DNAse treatment (Qiagen, Germany) was performed to remove genomic DNA. In the nuclear and cytoplasmic RNA, spike-in RNA extracted from rat liver (male Wistar) were added. Rat RNA was extracted from a mass of liver corresponding to 5% of the biopsie of the mouse liver. Polyadenylated (PolyA) nuclear and cytoplasmic RNA libraries were prepared with the TruSeq stranded mRNA LT kit (Illumina, San Diego, USA), starting from 650 ng of RNA, according to the manufacturer's protocol. Total RNA libraries (nuclear, cytoplasmic and unfractionnated) were prepared with the Kapa RNA hyperPrep with Riboerase prep combined with KAPA Unique Dual-Indexed Adapter Kit (Illumina, San Diego, USA), starting from 650 ng RNA. Libraries were sequenced on a Hiseq4000. We used a SBS 50 cycles and SR cluster kit, performing a single read sequencing of 65 nucleotides. We sequenced on average 70M reads for Nuclear PolyA and Nuclear Total samples,  25M reads for Cytoplasmic PolyA and Cytoplasmic Total samples, and 50M reads for the Unfractionated samples. 