title: GSM3768274: RNAseq_PCa_spheres_rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was purified with TRIzol reagent (Life Technologies). DNA contaminants were removed by treating RNA with 2U Turbo-DNaseI (Invitrogen) for 1h at 37°C and the RNA samples were re-purified using TRIzol. The quality of the isolated RNA was determined by Bioanalyzer 2100 (Agilent, Waldbronn, Germany). Only those samples with a 260nm/280nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5–2 were further processed. The TruSeq RNA Sample Prep Kit v2 (Illumina, Inc, California, USA) was used in the succeeding steps. Briefly, total RNA samples (100-1000ng) were poly A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples was fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the index for multiplexing Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Caliper GX LabChip® GX (Caliper Life Sciences, Inc., USA). The product is a smear with an average fragment size of approximately 260bp. The libraries were normalized to 10nM in Tris-Cl 10mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit HS4000 (Illumina, Inc, California, USA) was used for cluster generation using 10pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 2500 single end 100bp using the TruSeq SBS Kit HS2500 (Illumina, Inc, California, USA).
attributes.tag: GEO Accession
attributes.value: GSM3768274