GSM4263532: pullout_miR-28_bio_replicate_2; Homo sapiens; OTHER
Total RNA was extracted adding directly on adherent cells TRIzol reagent (Thermo Fisher) and following the instructions provided by the manufacturer. After DNAse treatment, 15 μg of RNA was incubated for 4 hrs at 4°C with 100 μl of streptavidin-conjugated beads (200 μl of Streptavidin Sepharose high performance, GE Healthcare) previously suspended in PO buffer (1M Tris pH8, 5M NaCl, 1M MgCl2, NP40 50 μl in 100 ml buffer). After 2 washes with PO buffer and 2 washes with DEPC-treated water, the RNA complexed with beads was recovered by adding 1 ml Trizol directly on the beads and then following the TRIzol RNA extraction protocol. The RNA isolated after the miRNA pullout procedure from both miR-28CT and miR-28BIO samples was used for the construction of the cDNA libraries using the TruSeq Stranded Total RNA Sample Preparation kit (Illumina) according to the manufacturer's suggestions. cDNA libraries were sequenced by HiSeq2000 (Illumina) in single-reads mode (50bp) by IGA Technology Service, Udine, Italy, obtaining about 20 million of reads for each samples.
GEO Accession
GSM4263532