title: GSM2107830: Neonatal_rep2; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated using RNeasy Mini Kit (Qiagen), and treated with DNase according to the manufacture’s protocol. Total RNA quality was evaluated using the 2100 Agilent Bioanalyzer and the RNA 6000 Nano kit: only samples with RIN score greater than 7 were further processed. RNA-Seq libraries were created using the Epicentre Script-Seq Complete Gold Kit-Low Input (Epicentre) composed of two modules: the Ribo-Zero Magnetic Kit depletion protocol for rRNA removal and the Script-Seq v2 Kit to prepare the libraries. 150 ng of total RNA was depleted and concentrated with RNeasy MinElute Cleanup Kit (Qiagen). Purified RNA was fragmented and first strand cDNA was synthesized using random hexamer with 5’ tagging sequences. After RNase digestion, a 3’ tag was introduced with random terminal tagging oligos. The obtained double tagged strand-specific cDNA was purified using AMPure XP beads (Beckman Coulter).  Libraries were enriched and indexed by 15 cycles of PCR amplification (ABI GeneAmp PCR system 9700) using FailSafe PCR enzyme and ScriptSeq Index PCR Primers for Illumina (Epicentre). After AMPure XP beads purification, library quality and size distribution were checked using an Agilent Bioanalyzer and DNA 1000 chip.  All libraries were quantified with a Qubit 2.0 Fluorometer (Life Technologies) using dsDNA BR Assay Kit (Life Technologies). RNA-Seq libraries were sequenced using TruSeq PE Cluster Kit v3 and TruSeq SBS Kit v3 (Illumina). According to index compatibility, the libraries were grouped into 2 pools and clustered on a TruSeq v3 flow cell at a concentration of 9pM to obtain > 50 million clusters for each sample. Paired end sequence reads (50-bp in length) were generated on an Illumina HiSeq 2000 (Samples 1 to 8)     Paired end sequence reads (75-bp in length) were generated on an Illumina NextSeq 500 (Samples 9 to 12)
attributes.tag: GEO Accession
attributes.value: GSM2107830