title: GSM2112494: RiboMinusRNAseq MCF7 normoxia, exp 3; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted using TriZOL according to manufacturers instructions, treated with Turbo DNase (Ambion) and repurified using RNeasy Mini Kits (Qiagen). Ribosomal RNA  was depleted from 5ug of total RNA using the RiboMinus Eukaryote System (Life technologies). cDNA synthesis was performed using SuperScript® III Reverse Transcriptase kit (Invitrogen). 3 µg of Random Primers (Invitrogen), 8 µL of 5× First-Strand Buffer and 10 µL of RNA mix was incubated at 94°C for 3 min and then at 4°C for 1 min. Then, 2 µL of 10 mM dNTP Mix (Invitrogen), 4 µL of 0.1 M DTT, 2 µL of SUPERase• In™ RNase Inhibitor 20U/ µL (Ambion), 2 µL of SuperScript™ III RT (200 units/µL) and 8 µL of Actinomycin D (1µg/µL) were added and the mix were incubated 5 min at 25°C, 60 min at 50°C and 15 min at 70°C to heat inactivating the reaction. The cDNA was purified by using 80 µL (2× volume) of Agencourt AMPure XP and eluted in 50 µL of the following mix: 5 µL of 10X NEBuffer 2, 1.5 µL of 10 mM dNTP mix (10mM dATP, dCTP, dGTP, dUTP, Sigma), 0.1µL of RNaseH (10 U/µL, Ambion), 2.5 µL of DNA Polymerase I Klenov (10U/µL, NEB) and water until 50 µL. The eluted cDNA was incubated for 30 min at 16°C, purified by Agencourt AMPure XP and eluted in 30 µL of dA-Tailing mix (2 µL of Klenow Fragment, 3 µL of 10X NEBNext dA-Tailing Reaction Buffer and 25 µL of water). After 30 min incubation at 37°C, the DNA was purified by Agencourt AMPure XP, eluted in TE buffer and quantified on NanoDrop. Libraries were subsequently prepared using the NextNEB Ultra library prep kit (NEB).
attributes.tag: GEO Accession
attributes.value: GSM2112494