GSM3099843: female young rep2; Homo sapiens; 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 14 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). Amplified cDNA was generated using the SMART-Seq v4 Ultra Low Input RNA kit (Clontech, Mountain View, CA).  Total RNA (10 ng) was used to synthesize first-strand cDNA utilizing proprietary template switching oligos.  Amplified double stranded cDNA is created by LD PCR using blocked PCR primers and unique sample barcodes were incorporated.  The result-ing cDNA was purified using AmpureXP beads (Beckman Coulter, Brea, CA).  Abundant Ribosomal cDNA was then depleted using R probes and 12 cycles of PCR with universal PCR primers completed the library.  The final libraries were purified using AmpureXP beads, and validated for appropriate size on a 4200 TapeStation D1000 Screentape (Agilent Technologies,, Santa Clara, CA).  The DNA libraries were quantitated using KAPA Biosystems qPCR kit and pooled together in an equimolar fashion.  Each pool was denatured and diluted to 16pM for On-Board Cluster Generation and sequencing on a HiSeq2500 sequencer for 100 paired-end sequencing following the manufac-turer's recommended protocol (Illumina Inc., San Diego, CA).
GEO Accession
GSM3099843