title: GSM2305916: rRNA depletion US-1492034; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was isolated from FFPE samples using the QIAGEN RNA Purification Kit and quantified using ribogreen assay. Quality of RNA was assessed using using Agilent RNA 6000 Nano Kit on a 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA) and the percentages of fragments larger than 200 nucleotides were calculated. RNA samples (100 ng total RNA) were fragmented at 94°C for 8 minutes on a thermal cycler. First strand cDNA syntheses were performed at 25°C for 10 minutes, 42°C for 15 minutes and 70°C for 15 minutes, using random hexameres and SuperScript II Reverse Transcriptase (Thermo Fisher Scientific Inc., Waltham, MA, USA). In a second strand cDNA synthesis the RNA templates were removed and a second replacement strand was generated by incorporation dUTP (in place of dTTP, to keep strand information) to generate ds cDNA. AMPure XP beads (Beckman Coulter, Inc., Indianapolis, IN, USA) were used to clean up the blunt-ended cDNA from the second strand reaction mix. The 3`ends of the cDNA were then adenylated to facilitate adaptor ligation in the next step. After ligation of indexing adaptors, AMPure XP beads were used to clean up the libraries. In a first PCR amplification step, PCR (15 cycles of 98°C for 10 seconds, 60°C for 30 seconds and 72°C for 30 seconds) were used to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. After validation of the libraries, using Agilent DNA 1000 kit on a 2100 Bioanalyzer instrument, the the libraries were quantitated by qPCR using KAPA Library Quantification Kit—Illumina/ABI Prism® (Kapa Biosystems, Inc., Wilmington, MA, USA) and validated using Agilent High Sensitivity DNA Kit on a Bioanalyzer. The size range of the DNA fragments were measured to be in the range of 200–650 bp and peaked around 250 bp.
attributes.tag: GEO Accession
attributes.value: GSM2305916