title: GSM1607539: Tfrc RIA sample; Mus musculus; RNA-Seq
library_construction_protocol: Cells were rinsed with PBS for 3 times and scraped off plates and centrifuged at 1500g for 5 minutes. Then pellets were lysed in lysis buffer (50mM Tris-HCl, pH7.0, 10mM EDTA, 1%SDS and freshly added DTT, phenylmethylsulphonyl fluoride (PMSF), Protease Inhibitor Cocktail Tablets). Lysate was incubated on ice for 20 minutes and sonicated by Covaris, and then diluted by two folds of hybridization buffer (500mM NaCl, 1%SDS, 100mM Tris-HCl, pH7.0, 10mMEDTA, 15%formamide, and freshly added DTT, PMSF, protease inhibitor, RNase-in). Antisense oligonucleotides for the target RNA were incubated with the lysate, rotating at 37 C overnight. Streptavidin beads were added to the hybridization buffer and incubated for 30 minutes at 37 C. Beads were accumulated by magnet rack and washed five times with wash buffer (2XSSC, 0.5%SDS, PMSF).RNA were eluted by 200ul RNA proteinase K buffer (100mMNaCl, 10mM TrisHCl, pH7.0, 1mM EDTA, 0.5%SDS, 1mg/ml proteinase K) and incubated at 50 C for 45 minutes, followed by 10 minutes boiling. RNA was purified by Trizol. Eluted RNA was treated by DNase and converted into cDNA by Ovation V2 kit (NuGEN), and subsequently converted into sequencing library using KAPA Library Preparation Kit.
attributes.tag: GEO Accession
attributes.value: GSM1607539