title: RNA-seq for siPRKDC and siControl in human HL-60/MX2 mitoxantrone resistant derivative of the HL-60 cell line
library_construction_protocol: PRKDC knockdown: 0.2X106 cells/mL were seeded per well in a 6-well plate and transfected the next day with siRNA at 80pmols PRKDC (siRNA select-ThermoFisher, 4390824) per well using the standard 6-well protocol for Lipofectamine3000 reagent (Invitrogen, L3000015). Cells were collected 48 h post-transfection and tested for the knockdown of the respective proteins by qPCR and immunoblotting. RNA was isolated from the cell pellet resuspended in TRIzol Reagent (Thermo Fisher Scientific) using the chloroform-isopropanol method. The RNA samples were quantitated on a Qubit fluorometer and appropriate dilutions were loaded on a high-sensitivity RNA screen tape to determine the RNA integrity number (RIN). Samples with 28S/18S peak integrated area ratios >2 were used for library preparation. 1.5 μg of QC verified RNA samples was used for poly (A) mRNA isolation from total RNA using NEB Next Poly (A) mRNA Kit (Cat. no. NEB# 7490) according to the manufacturer's protocol. The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat. no. E7760L) was used to construct double-stranded cDNA libraries from the polyA RNA fraction, using RNA fragmentation, first strand cDNA synthesis with random primers, second strand cDNA synthesis, end repair of double-stranded cDNA, adapter ligation, removal of excess adapter using sample purification beads, PCR enrichment of adapter ligated DNA, and clean-up of PCR products as instructed by the manufacturer. The cleaned libraries were quantitated on a Qubit fluorometer and appropriate dilutions were loaded on a high sensitivity D1000 screen tape to determine the size range of the fragments and the average library size.