NextSeq 500 paired end sequencing; RNA-seq of human liver cancer cell lines after RBP-KD
RNA-seq of human liver cancer cell lines after RBP-KD
HepG2 and Huh7 human hepatocellular carcinoma cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD). All cell lines were mycoplasma free when periodically tested with Mycoplasmacheck (Eurofins Genomics). Huh7 and HepG2 cells were cultured in T75 flasks at 37 °C and 5 % CO2 atmosphere using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1/100 Penicillin/Streptomycin (P/S, Sigma) and 10 % fetal bovine serum (Hyclone, GE healthcare). Huh7 cells were maintained by splitting 1/6 three times a week, HepG2 by splitting 1/4 three times a week. This was done by aspirating the medium, gently washing the cells with phosphate buffered saline (PBS, without Mg2+, Sigma) and detaching them with 2 mL of a trypsin-EDTA solution (0.05%, Sigma) for 3-5 min. Trypsin-EDTA was inactivated with a minimum of 10-fold surplus of culture medium before a cell fraction was passaged. At 70-80% confluency, cells were harvested by trypsination and subjected to electroporation. Small interfering RNA (siRNA) POOLs targeting PEG10, HIST1H1C, KPNA2, PKM, NQO1, TOP2A, CCT3, STMN1, DDX39A, and IGF2BP1 was used (Thermo Scientific). siRNA ON-TARGETplus Non-Targeting siRNA#1 (Thermo Scientific) was used as control. Cells were washed twice in PBS and once in OptiMEM without phenol red (Invitrogen). A total of 5 µg siRNA was transferred to a 4-mm cuvette (Bio-Rad), and 5 million cells were added in 200 μl OptiMEM and incubated for 3 min before being pulsed with an exponential decay pulse at 300 V, 250 μF, in a Genepulser II (Bio-Rad). Immediately after electroporation, the cells were transferred to pre-heated (37˚C) phenol red-free DMEM culture medium supplemented with 10% (v/v) FBS and no antibiotics.  Upon cell harvest, 700 µL Qiazol (QIAGEN) was directly added onto the cells on ice and mixed. The cell extract was either stored at -80 °C or RNA extraction was continued immediately by adding 140 µL chloroform. This Qiazol/chloroform mixture was shaken for 30 sec and incubated at RT for 3 min, before centrifugation at 9.000 g for 5 min at 4 °C. The upper aqueous phase was transferred to a new tube and an equal volume of isopropanol was added. The tube was inverted 5 times followed by 10 min incubation at RT. The mixture was centrifuged at 9.000 g and 4 °C for 10 min and the supernatant discarded. The pellet was washed in 700 µL cold 70% ethanol and centrifuged at 15.000 g for 5 min at 4 °C. The supernatant was discarded entirely, and the pellet air-dried for 5 minutes, before resuspension in nuclease-free water. RNA concentration was determined by nanodrop (Nanodrop 2000c). RNA was DNase-treated using the Turbo DNase Kit (Thermo Fisher) for 30 min at 37 °C and the reaction was stopped by adding the inactivation reagent from the kit and incubation for 5 min at 37 °C (Thermo Fisher). In order to achieve higher purity, the RNA was EtOH-precipitated overnight using 1/10 of 3M sodium acetate (pH 5.2) and 1 µL GlycoBlue (Ambion), followed by washing twice with 700 µL 70 % ethanol. The pellet was air-dried, resuspended in 25 µL nuclease-free water and the RNA concentration determined by nanodrop and purity (RIN) by Bioanalyzer. The library preparation was carried out using the Illumina TruSeq® Stranded RNA Library Prep Kit v2 (dual index) according to the manufacturer's instruction and ribosomal depletion was achieved with Ribo-Zero Gold. The quality of every cDNA library was determined on an Agilent Bioanalyzer instrument according to the manufacturer's protocol. All libraries were quantified with the KAPA-SYBR FAST qPCR kit (Roche).
Experimental Factor: RNA interference
PEG10 knockdown by siRNA
Experimental Factor: cell line
HepG2