GSM4041565: RNA-6; Mus musculus; RNA-Seq
The purification of total and RNA was performed using the miRNeasy micro kit (Qiagen, Germantown, MD).  Flow sorted cells were homogenized with the addition of 700 ul of QIAzol Lysis Reagent. After addition of chloroform, the homogenate was separated into aqueous and organic phases by centrifugation.  The upper, aqueous phase was extracted.  On-column DNAse digestion was also performed to remove any residual genomic DNA contamination followed by additional washes.  High quality RNA was then eluted in 18 ul of RNase-free water.  Quantitative assessment of the purified total RNA was accomplished by using a Qubit Broad Range RNA kit (Thermo Fisher, Waltham, MA).  The RNA was further evaluated qualitatively by a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Sequencing libraries were prepared using the TrueSeq Stranded mRNA Kits (Illumina Inc., San Diego, CA) according to the manufacturer's instructions. Sequencing was performed using NextSeq 500/550 v2.5 sequencing reagent kits and The NextSeq 550Dx instrument (Illumina Inc.) Paired-end RNA-Seq