title: GSM2796085: Notch1_proximal_aorta_3; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated using a combination of Trizol and Total RNA Purification Kit (Norgen Biotek). Twelve (12) total RNA samples from mouse aorta tissues (n=3 of each group), were submitted to ORB for mRNA-Sequencing. The RNA was digested with RNase free DNase I (Epicentre; Part # D9905K) and re-purified on RNeasy MinElute columns (Qiagen; Part #: 74204) using an alternative high-ethanol binding condition to preserve Low Molecular Weight (LMW) RNAs. The newly prepared digested total RNA samples were then quantified by Quant-iT RiboGreen RNA fluorometric assay (Thermo Fisher Scientific; Part # R11491) and assessed for quality by Agilent2100 Bioanalyzer RNA 6000 Pico Assay (Agilent Technologies; Part # 5067-1513). Amplified cDNA was prepared from a total of 10 nanograms (ng) of the digested total RNA samples using the NuGen Ovation RNA-Seq System v2 (NuGen: Part # 7102-A01) and fragmented using NEBNext dsDNA Fragmentase (NEB: Part # M0348L). Template DNA molecules suitable for cluster generation were then prepared from the fragmented cDNA using the Illumina TruSeq DNA Nano HT Library Preparation Kit (Illumina Inc.: Part # FC-121-4003). The quality and size distribution of the amplified cDNA libraries were determined by chip-based capillary electrophoresis using an Agilent 2100 Bioanalyzer DNA 1000 Chip assay (Agilent Technologies: Part # 5067-1504). The libraries were then quantified using the KAPA Library Quantification Kit (Kapa Biosystems; Boston MA).  The libraries were pooled at equimolar concentrations and diluted prior to loading onto the flow cell of the Illumina cBot cluster station. The libraries were extended and bridge amplified to create sequence clusters using the Illumina HiSeq PE Cluster Kit v4 and sequenced on an Illumina HiSeq Flow Cell v4 with 50-bp paired-end reads plus dual index read using the Illumina HiSeq SBS Kit v4. Real time image analysis and base calling were performed on the HiSeq2500 using the HiSeq Sequencing Control Software version 2.2.68.
attributes.tag: GEO Accession
attributes.value: GSM2796085