GSM5465261: 293T_KuDNA-PKcs+/- + siControl_rep1; Homo sapiens; RNA-Seq
Total RNA from all samples (3 repeats for each coditions) was extracted using TRIzol reagent (Life Technologies) following the manufacturer's protocol 72h after siRNA transfection. 1 µg of total RNA was fragmented by incubation at 900C for 6 min in 40 µL of 100 mM Tris-HCl pH 8.0 and 8 mM MgCl2. After isopropanol precipitation RNA was dissolved in 12 µL and subjected to ribosomal RNA depletion by NEBNext rRNA depletion kit (E6310L, New England Biolabs) according manufacturer's protocol. mRNA sequencing libraries were prepared by NEBNext Ultra II Directional RNA library Prep kit for Illumina (E7760S, New England Biolabs) according to the manufacturer's protocol. NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set1) (E7600S, New England Biolabs) were used for adaptor ligation. Quality control of the libraries was carried out on Agilent 2100 Bioanalyzer (Agilent Technologies). RNA libraries were prepared for sequencing using standard Illumina protocols