GSM4232791: DDX6 CLIP-seq + DENV2 Rep2; Homo sapiens; RNA-Seq
HuH-7 cells with or without DENV-2 infection (MOI = 5) were first irradiated by UV254 nm at 400 mJ/cm2 and lysed prior to perform DNase I treatment (NEB, #M0303L) at 37°C for 5 min. Cell lysates were clarified by centrifugation and performed DDX6 pull-down at 4°C for overnight. The RNA fragmentation was performed directly on beads by utilizing 4 U of ShortCut® RNase III (NEB, #M0245S) at 37°C for 4 min. Subsequently, 3´ end of shortened RNA was ligated with 5´ pre-adenylated linkers using T4 RNA ligase 2, truncated KQ (NEB, #M0373L) activity. For excess linkers removal, deadenylase (NEB, #M0331S)] and Terminator™ 5´-Phosphate-Dependent Exonuclease (Epicentre, #TER51020)] were used. Proteinase K (NEB, #P8107S) and Quick-RNA™ MicroPrep (Zymo Research, #R1051) were utilized to extracted the RNAs. Reverse transcription (RT) with SuperScript™ IV Reverse Transcriptase (Thermo Fisher Scientific, 18090050) was performed at 42°C for 20 min and 50°C for 20 min. The cDNAs were cleaned and poly-A tailed by exonuclease I (NEB, #M0293S) and terminal transferase (NEB, #M0315S), respectively. cDNA libraries were amplified with specific primers containing Illumina compatible sequences, and KAPA HiFi HotStart DNA polymerase (KAPA Biosystems, #KK2501) at 98°C for 3 min, 40°C for 1 min and 72°C for 1 min. The size of libraries that larger than 130 bp of self-annealing primers, were further purified in 2 steps by using QIAquick PCR purification kit (QIAGEN, #28106) and AMPure® XP magnetic beads (Beckman Coulter, #A63881), respectively. The concentration and size of cDNA libraries were verified by utilizing Qubit™ fluorometric quantitation [Quant-iT™ PicoGreen® dsDNA Reagent and Kits (Thermo Fisher Scientific, #P7589)] and Bioanalyzer. Finally, cDNA libraries were performed paired-end sequencing on Illumina HiSeq 4000.
GEO Accession
GSM4232791