title: GSM1668999: R273HT-mutant-damage-induced-Replicate1; Homo sapiens; miRNA-Seq
library_construction_protocol: Small RNA fractions from cells were isolated using PureLink miRNA Isolation kit (Ambion, USA) according to the manufacturer’s protocol. Briefly, in a two-column based purification method, cells were first resuspended in binding buffer containing guanidine isothiocyanate and mixed with ethanol (final concentration of 35%). The mix was then passed through a spin column and flow through containing small RNAs was collected. Ethanol (final concentration 70%) was added to the flow through fraction and the mix was processed through a silica based column to bind the small RNAs to the silica membrane. The impurities were subsequently removed by washing the column with wash buffer and small RNAs were eluted in RNase free water. Total RNA-Seq kit V2.0 (Life Technologies, Thermo Fisher Scientific Inc., MA USA) was used to prepare small RNA cDNA libraries. Briefly, small RNAs (~150-250 ng) were first mixed with Hybridization Solution and Ion Adaptor Mix v2 and hybridization reaction was performed in thermal cycler. Ligation enzyme mix was then added to the hybridization reactions and incubated in a thermal cycler at 16°C for 16 hours. Adapter ligated small RNA samples were subsequently reverse transcribed with Ion RT Primer v2 and SuperScript® III Enzyme Mix to make cDNAs. The cDNA products were then purified and size-selected using the magnetic bead cleanup module and PCR amplified with Ion 5’ and 3’ PCR Primers. The amplified DNAs were quantified and assessed for size distribution using Agilent® DNA 1000 Kit in Agilent® 2100 Bioanalyzer.   Each cDNA library was then clonally amplified on Ion Sphere™ Particles (ISPs) by emulsion PCR in Ion OneTouch™ System using Ion OneTouch™ 200 Template Kit v2 DL (Life Technologies, Thermo Fisher Scientific Inc., MA USA). Enrichment of the template positive ion spheres was carried out in Ion OneTouch™ Enrichment System. Template enriched ion sphere particles were subsequently sequenced on the Ion PGM™ sequencer using the Ion PGM™ 200 Sequencing Kit ((Life Technologies, Thermo Fisher Scientific Inc., MA USA) and Ion 316™ Chip.
attributes.tag: GEO Accession
attributes.value: GSM1668999