GSM4263620: control embryos replicate 5; Bos taurus; RNA-Seq
Total RNA from these 21 embryo pool samples was isolated using the RNeasy Micro kit (QIAGEN) according to the manufacturer's instructions. RNA quality and concentration were analyzed using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA.), NanoDrop (ThermoFisher) and Quantus Quantiflour® RNA system (Promega). Samples with best RNA quality and concentration were selected for generation of RNA-Seq libraries (15 libraries, 5 replicates/embryo treatment). For library preparation, the Ovation SOLO RNA-Seq System Kit from NUGEN Technologies was used. Library preparation followed the manufacturer's instructions. In brief, aliquots of 1 ng of total RNA were prepared for each embryo sample as starting material for RNA-Seq library preparation. First, samples were subjected to DNase treatment and primer annealing, followed by cDNA processing and second strand synthesis. After end repair, adapters were ligated and the first step of library amplification was performed using a number of PCR cycles based on the results of the library amplification optimization step by qPCR. To remove fragments derived from ribosomal RNAs, NuGEN's insert-dependent adaptor cleavage (InDA-C) technology was performed in the next step. At the same time, strand selection is performed. After this step, the second library amplification and purification were performed for each sample using a universal primer and a set of barcode primers for sample multiplexing. Once RNA-seq libraries were prepared, quantitative and qualitative analyses were performed for each of the libraries using Agilent Bioanalyzer 2100 DNA High Sensitivity assays and Quantus Quantiflour® ONE dsDNA system (Promega). low-input total RNA-Seq
GEO Accession
GSM4263620