GSM5315741: C166_24hr_nuc_2; Mus musculus; RNA-Seq
Cells were fractionated into nuclear and cytoplasmic fractions and RNA isolated according to a protocol by Gagnon et al. (Gagnon K, Li L, Janowski BA, Corey DR. Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading. Nature Protocols 2014, vol. 9, no. 9, p.2045-60). The library preparation was done using TruSeq® stranded total RNA sample preparation kit with rRNA depletion (Illumina inc).The starting material (300 ng) of total RNA was rRNA depleted using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal. The isolated mRNA was subsequently fragmented using enzymatic fragmentation. Then first strand synthesis and second strand synthesis were performed and the double stranded cDNA was purified (AMPure XP, Beckman Coulter). The cDNA was end repaired, 3' adenylated and Illumina sequencing adaptors ligated onto the fragments ends, and the library was purified (AMPure XP). The mRNA stranded libraries were pre-amplified with PCR and purified (AMPure XP). The libraries size distribution was validated and quality inspected on a Bioanalyzer high sensitivity DNA chip (Agilent Technologies). High quality libraries were quantified using qPCR, the concentration normalized and the samples pooled according to the project specification (number of reads). The library pool(s) were re-quantified with qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing on HiSeq2500) instrument using hiSeq v.4 reagents (51 cycles) (Illumina Inc.).(service from Exiqon A/S, Vedbaek, Denmark)