GSM1668430: SiR3ET (mRNA); Homo sapiens; RNA-Seq
RNA was prepared using RNA/protein spin columns (Clontech, Mountain View, CA).  Lysis solution was added to cells in 6-well plates and cells were solubilized by passage of tissue successively through 21 and 27 gauge needles.  Individual cell lysates were applied to RNA spin columns for purification.  RNAs eluted into nuclease-free water were quantitated by UV spectroscopy, quality assessed by 260nm/280nm ratio then frozen at -80 0C.  Before library construction, RNA quality was assessed by capillary electrophoresis against a known size standard. Poly-A containing mRNA was purified using poly-T oligo attached magnetic beads. The purified mRNA was fragmented and ethanol precipitated. The RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Second strand cDNA synthesis was performed using DNA polymerase I and RNase H. The cDNA was purified ends were repaired and phosphorylated using Klenow, T4 polymerase, and T4 polynucleotide kinase, after which an “A” base was added to the 3' ends of double-stranded DNA using Klenow exo- (3' to 5' exo minus).  Paired end DNA adaptors (Illumina) with a single “T” base overhang at the 3' end were ligated. DNA fragments of approximately 250-300 bp were purified on, and excised from agarose gels.  The adapter-modified DNA fragments were enriched by PCR using primers PE 1.0 and PE 2.0 (Illumina). The concentration and size distribution of the libraries was determined on an Agilent Bioanalyzer DNA 1000 chip. mRNA libraries were loaded onto Illumina TruSeq v3 paired end flow cells at concentrations of 9 pM to generate cluster densities of 600,000-800,000/mm2 following Illumina's standard protocol using the Illumina cBot and TruSeq Rapid Paired end cluster kit version 3.  The flow cells were sequenced as 51 X 2 paired end reads on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3 and HiSeq data collection version 2.0.12.0 software. Base-calling is performed using Illumina's RTA version 1.17.21.3.
GEO Accession
GSM1668430