title: NextSeq 500 paired end sequencing; RNA-seq in HAP1 tRNA gene locus deleted clones and control clones
design: RNA-seq in HAP1 tRNA gene locus deleted clones and control clones
library_construction_protocol: Cells were harvested at 80% to 90% confluency. 1,000,000-5,000,000 cells were pelleted and lysed in 700 μL Qiazol (QIAGEN). 140 μL chloroform were added. The mixture was shaken for 30 sec and incubated for 2.5 min at room temperature, followed by centrifugation at 9,000×g for 5 min at 4°C. The upper aqueous phase was mixed with 1 volume isopropanol thoroughly, followed by 10 min incubation at room temperature. The RNA was pelleted by centrifugation at 9,000×g for 10 min at 4°C and washed once with ice-cold 70 % ethanol. RNA concentration and purity were determined by nanodrop (Nanodrop 2000c). To remove genomic DNA, 10 μg RNA were mixed with 5 μL 10×TurboDNase buffer (Invitrogen), 1 μL TurboDNase (Invitrogen), 1 μL RNase Inhibitor (RiboLock, Invitrogen), and water was added to a total volume of 50 μL. The samples were incubated at 37°C for 30 min and purified with the Zymo RNA Clean & Concentrator Kit (Zymo Research). The concentration of purified RNA was determined by Qubit, and the integrity was assessed on an Agilent Bioanalyzer instrument with RNA 6000 Nano kit according to the manufacturer's protocol. The RNA was first enriched for molecules with PolyA tail with NEBNext® Poly(A) mRNA Magnetic Isolation Module. The RNA library preparation was performed using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina according to the manufacturer's instruction.