GSM5088697: HeLa-tet rep 2; Homo sapiens; RNA-Seq
HeLa cells, HeLa-tet and HeLa-tet-TgNSM-TY cell lines were seed at a density of 0.06x10e6 /ml in T25 flasks in presence of DOX 1µg/ml. After 48 hr culture media was replaced for fresh DOX media ±IFNγ 100U/ml. Cells were collected 6 hr post ±IFNγ media swap and their RNA was extracted and purified from using the RNeasy kit (QIAGEN). Total RNA from three independent biological replicates was submitted to the Genome Access Technology Center (Washington University School of Medicine) for library prep and sequencing. Total RNA integrity was determined using Agilent Bioanalyzer. Library preparation was performed with 5 to 10ug of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. For bradyzoite and HeLa cells experimnts fragments were sequenced on an Illumina illumina NovaSeq 6000 using paired-end reads extending 150 bases.