title: GSM3474064: dermis, 1month, Control rep2; Mus musculus; RNA-Seq
library_construction_protocol: FACS sorted populations were pelleted at 12,000g, and then RNA extracted using the QIagen RNAeasy Fibrous tissue kit as per the manufacturers instructions, including optional on column DNAse. RNA was then initially quantified by nanodrop, followed by quantification using capillary cassette. RNA-Seq libraries were prepared using the illumina TruSeq Stranded Total RNA LT (Ribo-Zero Gold) Sample Prep Kit (illumina, RS-122-2301/RS-122-2302), according to the standard manufacturer's protocol (illumina, 15031048 Rev. E October 2013) described briefly as follows. To enrich for mRNA, up to 100 ng of total RNA (range of 34-100 ng) was depleted of rRNA using Ribo-Zero Gold. The enriched mRNA was then subjected to a heat fragmentation step aimed at producing fragments between 120-210 bp (average 155 bp). cDNA was synthesized from the fragmented RNA using SuperScript II Reverse Transcriptase (Invitrogen, 18064014) and random primers. The resulting cDNA was converted into dsDNA in the presence of dUTP to prevent subsequent amplification of the second strand and thus maintaining the 'strandedness' of the library. Following 3' adenylation and adaptor ligation, libraries were subjected to 15 cycles of PCR to produce libraries ready for sequencing. The libraries were quantified on the Agilent BioAnalyzer 2100 using the High Sensitivity DNA Kit (Agilent, 5067-4626). Libraries were pooled in equimolar ratios, and the pools were quantified by qPCR using the KAPA Library Quantification Kit - illumina/Universal (KAPA Biosystems, KK4824) in combination with the Life Technologies Viia 7 real time PCR instrument. Sequencing was performed using the Illumina NextSeq500 (NextSeq control software v2.0.2 / Real Time Analysis v2.4.11). The library pool was diluted and denatured according to the standard NextSeq protocol (Document # 15048776 v02), and sequenced to generate single-end 76 bp reads using a 75 cycle NextSeq500/550 High Output reagent Kit v2 (illumina, FC-404-2005). Library preparation and sequencing was performed at the Institute for Molecular Bioscience Sequencing Facility (University of Queensland).
attributes.tag: GEO Accession
attributes.value: GSM3474064