Illumina HiSeq 2500 paired end sequencing; Quantifying the transcriptional effects of lncRNA depletion in HeLa cells with RNA-seq
Quantifying the transcriptional effects of lncRNA depletion in HeLa cells with RNA-seq
HeLa cells were obtained from American Type Culture Collection. HeLa cells were maintained in Dulbecco's modified Eagle's medium (Sigma Aldrich, D6429) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and cultured at 37 degrees Celsius with with 5% CO2. HeLa cells were transfected with Lipofectamine RNAiMax reagent (Thermo Fischer Scientific) following the manufacturer's instructions, using siRNAs (Thermo Fischer Scientifc) and LNA Gapmers (Exiqon) at a final concentration of 50 nM and 25nm, respectively. All experiments were done 48 hr after transfection. The QuantiTect Reverse Transcription Kit (QIAGEN) was used for cDNA synthesis including an additional step to eliminate genomic DNA contamination. RNA (1 ug) was extracted with the RNeasy Kit (QIAGEN) and treated with DNase I following the manufacturer's instructions. RNA quality was assessed using a Total RNA Nano chip with a 2100 Bioanalyzer instrument (Agilent). RNA-seq libraries were prepared from HeLA cells using TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (Illumina, RS-122-2303). Library quality was assessed using a DNA1000 chip with a 2100 Bioanalyzer instrument (Agilent).
Experimental Factor: loss of function method
RNA interference
Experimental Factor: compound
Ambion control