NextSeq 500 paired end sequencing; Retinitis pigmentosa-linked mutation in DHX38 modulates its splicing activity
RNA was isolated directly from cultured HEK293 cells. HEK293 cells were cultured in high-glucose (4500 mg/l) DMEM medium (Sigma-Aldrich) supplemented with 10% foetal bovine serum, 1% penicillin and streptomycin. Cells were grown in 37 C and 5% CO2 atmosphere. 50nM siRNA SMART Pool against DHX38 50nM was transfected into cells using Oligofectamine reagent (Invitrogen) at the final concentration of 50nM according to the manufacturers protocol. The negative control # 5 siRNA from Ambion was used as a negative control. Total RNA was isolated from harvested cells using TRIzol followed by Direct-zol RNA MicroPrep (Zymo Research). Libraries were prepared using KAPA RNA Hyperprep Kit with RiboErase (Kapa Biosystems/Roche).