title: GSM2943841: TCIPP 25uM rep1; Homo sapiens; RNA-Seq
library_construction_protocol: To ensure for high quality and good yield of RNA, the combined TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy (Qiagen, Hilden, Germany) approach was adopted. Briefly, this involved lysing cells and extracting total RNA by phase separation using TRIzol as per the manufacturer's instructions. The aqueous phase containing the crude total RNA extraction was then subjected to clean up using the RNeasy kit according to the manufacturer's instructions, resulting in pure total RNA. The TruSeq Stranded mRNA Sample Preparation kit (Illumina, San Diego, CA, USA) was used following the manufacturer's protocol to construct sequencing libraries. Briefly, 4 μg total RNA was subjected to purification using poly-T oligo-attached magnetic beads to purify for poly-A containing mRNA. mRNA was then fragmented using divalent cations and exposure to high temperatures. Fragments were then reverse transcribed into first-strand cDNA using random hexamer primers followed by second-strand cDNA synthesis to create blunt-ended, double-stranded cDNA. Unconverted RNA was then depleted by RNase H incubation. Fragment 3′ ends were then adenylated to prevent ligation to one another during adaptor ligation. Successful adaptor-ligated fragments were purified and enriched by means of PCR using primers that annealed to adaptors to create the final cDNA library. Constructed sequencing libraries were then paired-end sequenced (2 × 50 bp) using the Illumina HiSeq 1500 platform (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM2943841