title: GSM1937106: ARD-07_5_S1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA were extracted from tissues and cell pellets using the RNeasy mini Kit (Qiagen), quantified using a NanoDrop™ 8000 spectrophotometer (Thermo Scientific) and quality controlled using a 2100 Electrophoresis Bioanalyzer (Agilent). Libraries of template molecules suitable for strand specific high throughput DNA sequencing were created using “TruSeq Stranded Total RNA with Ribo-Zero Gold Prep Kit” (catalog # RS-122-2301, Illumina Inc.). Briefly, the removal of cytoplasmic and mitochondrial ribosomal RNA (rRNA) was performed from 500 ng of total RNA using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, the RNA was fragmented using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single ‘A’ nucleotide was added to the 3’ ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C). Excess of PCR primers was removed by purification using AMPure XP beads (Agencourt Biosciences Corporation). Final cDNA libraries were quality-checked and quantified using a 2100 Electrophoresis Bioanalyzer (Agilent). The libraries were loaded in the flow cell at 7pM concentration and clusters were generated in the Cbot and sequenced in the Illumina Hiseq 2500 as paired-end 2x50 base reads following Illumina's instructions.
attributes.tag: GEO Accession
attributes.value: GSM1937106