GSM4729154: SUM52-K599A; Homo sapiens; RNA-Seq
Total RNA was extracted with an RNeasy Plus Mini Kit in accordance with the manufacturer's protocol (Qiagen, Hilden, Germany). RNAs from SUM52 cells transducted with the IRE1 dominant-negative K599A, K907A, or control adenovirus were equally pooled (total 3 μg) and sequenced by LC Sciences. RNA was subjected to isolated poly(A) mRNA with poly-T oligo-attached magnetic beads (Invitrogen). Following purification, the poly(A)- or poly(A)+ RNA fractions were fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA). Approximately 1 µg of this RNA was prepared for Next Generation Sequencing using a version of the Illumina protocol, Directional_mRNA-Seq_SamplePrep_Guide_15018460_A.