GSM2973528: 01W_C_02; Rattus norvegicus; RNA-Seq
RNA was extracted using the Nucleospin RNA XS kit (Macherey-Nagel, Germany). Briefly, tissue samples were lysed in 1 ml TRIzol reagent (Thermo Fisher Scientific, USA) and mechanically disrupted using a homogenizer. Chloroform was added and the aqueous  phase containing the RNA was added onto the Nucleospin column. From there the manufacturer's protocol was followed including the DNAse treatment. The quality and quantity of the isolated total RNA was determined with a Qubit 2.0 Fluorometer (Life Technologies,  USA) and a Tapestation 2200 (Agilent, Germany) cDNA libraries were prepared by the Functional Genomics Center Zürich (University and ETH Zürich) according to the procedure described as follows: TruSeq Stranded Total RNA Library Prep Kit (Illumina Inc, USA) was used in the following steps. Briefly, the 200 ng total RNA samples were ribo-depleted using Ribo Zero Gold (Illumina Inc, USA) and fragmented with divalent cations and an elevated temperature. The fragmented RNA was then reverse-transcribed into cDNA with Actinomycin, which prevents DNA-dependent synthesis but allows RNA-dependent synthesis. Actinomycin is added during first strand synthesis. To achieve strand specificity, dUTP was incorporated instead of dTTP in the course of the second strand synthesis. The polymerase used for the subsequent PCR amplification will not incorporate past dUTP and so the second strand will be suppressed. A single 'A' nucleotide is added to the 3' ends of the cDNA fragments before ligation of the TruSeq adapters. The adapters contain the index for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using qPCR (LightCycler 96, Roche, Switzerland) and Tapestation 2200 (Agilent, Germany). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20.    Cluster Generation and sequencing were performed by the Functional Genomics Center Zürich (University and ETH Zürich) according to the procedure described as follows: The TruSeq SR Cluster Kit v4-cBot-HS (Illumina Inc, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 4000 single end 125 bp using the TruSeq SBS Kit v4-HS (Illumina Inc, USA).