title: GSM1290670: liver_mRNA_bowtie RNA-Seq; Mus musculus; RNA-Seq
library_construction_protocol: Embryos were depleted of extraembryonic tissues using glass needles, harvested into TriZol and snap frozen. RNA was isolated using TriZol and the PureLink™ RNA Micro Kit (Invitrogen), following the manufacturer's instructions. Integrity of the RNA samples (Table S1) was confirmed using the Experion Automated Electrophoresis Station (2286; BioRad). Concentration of the RNA samples was determined by a Qubit fluorometer (Invitrogen; Q32857) and the Experion. In order to generate RNA-Seq expression profiles from the early embryonic stages, the Ovation RNA-Seq kit (Nugen) and the SMARTer Ultra Low RNA kit from Illumina were tested for low quantity RNA-Seq expression profiling on mouse liver RNA. The results were compared to regular RNA-Seq profiling. Regular RNA-Seq profiling was performed on 100 ng of RNA, using the RiboZero kit (EpiBio) to deplete for ribosomal RNA, and hexamers for priming the reverse transcription, followed by regular sample preparation for sequencing. 100 pg of mouse liver total RNA was amplified using the Ovation RNA-Seq kit (7100-08). cDNA was sonicated using the Bioruptor (Diagenode) for 15 min (30 sec on, 30 sec off, high power). Paired-end sequence adaptors were ligated to DNA fragments, followed by size selection (300 bp; E-Gel® Agarose Gel Electrophoresis System of Invitrogen) and 14 cycles of PCR amplification. For the SMARTer Ultra Low RNA kit from Illumina, 100 pg of mouse liver total RNA was amplified as described below. Sequencing was performed as described below. Direct comparisons using RNA-Seq sequencing showed that the SMARTer Ultra Low RNA was clearly superior and this was chosen to be the profiling method of choice. For all samples, 100 pg of RNA was used for cDNA synthesis and amplification using the SMARTer™ Ultra Low RNA Kit for Illumina Sequencing (Clontech; Cat. No. 634935) using 20 PCR cycles. The amplified cDNA was fragmented by sonication (Bioruptor, Diagenode). Paired-end sequence adaptors were ligated to DNA fragments, followed by size selection (300 bp; E-Gel® Agarose Gel Electrophoresis System of Invitrogen) and 14 cycles of PCR amplification.
attributes.tag: GEO Accession
attributes.value: GSM1290670