title: GSM9022539: control 4; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted using the TRIzol reagent according to the manufacturer's protocol. 1 ug of total RNA was used for the construction of sequencing libraries. Then the libraries were constructed using TruSeq Stranded mRNALTSample Prep Kit(Ilumina,San Diego, CA, USA) .RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) . RNA concentration was measured using Qubit® RNA Assay Kit in Qubit®2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations.  Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample.  In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq6000 platform and 150 bp paired-end reads were generated.