GSM1153068
GSM1153068: MCF7_Hypoxia_48h_microRNAseq_rep1; Homo sapiens; RNA-Seq
RNA was extracted from cells using the miRVana miRNA isolation kit (Ambion) in accordance with the prescribed protocol provided with the kit. RNA quality was assessed after extraction using Agilent bioanalyser. Libraries were prepared from the 20 to 30 nucleotide RNA fraction according to the Illumina DGE-small RNA sample preparation protocol (v1.5). The 20 to 30 nucleotide RNA fraction was isolated from 2µg of total RNA after being run in a 15% urea-TBE gel (Invitrogen) for 1h at 200V. The RNA contained on the excised band was eluted in 300µl of 0.3M NaCl by 4h of constant rotation at room temperature. The elute was separated from the gel debris using a Spin-X-column (Thermo Fisher Scientific) and RNA was precipitated by adding 750µl of 100% ethanol and 3µl of glycogen (1mg/ml) and incubating for 30 minutes at -80˚C. The precipitated RNA was centrifuged at 14,000 rpm for 25 minutes at 4˚C, washed with 75% ethanol, air dried and resuspended in 5µl of RNAse-free water. This material was used to prepare miRNA sequencing libraries using the DGE-small RNA sample preparation kit (v1.5) from Illumina and according to the protocol provided with the kit.
GEO
GSM1153068
GEO Accession
GSM1153068