title: K-562_K700E_cyto_RNA_2
organism: Homo sapiens
description: Protocols: 10^7 cells were harvested by centrifugation at 300 x g for 5 minutes before fractionation and RNA extraction RPMI1640 w/ 2mM L-Glutamine (Sigma-Aldrich) & 10% FBS Superior (Biochrom) & 1mM Ultra-Glutamine (Lonza) & 1% P/S (Gibco) Cytoplasmic/Nuclear RNA extraction was performed essentially as described in Gagnon et al. Nat. Protocols 2014. Briefly, cells were resuspended in ice-cold hypotonic lysis buffer (HLB) supplemented with SUPERase Inhibitor (Ambion). The free nuclei were then precipitated by centrifugation at 1000 x g for 3 minutes. The cytoplasmic supernatant was spun again at maximum speed for 2 minutes to remove any contaminating nuclei. Nuclei and cytoplasmic fractions were mixed directly with TRIzol supplemented with 1mM EDTA to inhibit ion-mediated RNA catalysis and heated at 65°C for 10 minutes. Samples were mixed vigorously with chloroform and spun at maximum speed at 4°C for 15 minutes. The aqueous, RNA-containing, fraction was then mixed 1:1 with ethanol and applied to an RNA-binding column (Direct-zol™ R2051, Zymo Research). The RNA was then washed and on-column DNAse I digested, before elution with RNase-free water. Library preparation used the SMARTer Stranded Total RNA Sample Prep Kit from Takara Clontech (Cat. No. 634873) and a 2% by mass RNA spike-in of Lexogen Spike-In RNA Variants (SIRV 025.03) was included.