GSM5740334: RATE-seq Neu WT SS rep1; Homo sapiens; RNA-Seq
After 4sU incorporation, cells were quickly washed twice with ice-cold 1× PBS Total RNA and scraped into ice-cold 1.5 ml Eppendorf tubes.  Cells were collected by spinning at 1000g for 5 min at 4°C and cell pellets were resuspended in 1 mL of Trizol (Thermo Fisher Scientific). Total RNA was extracted according to manufacturer instructions. The SS sample was prepared from a 5μg aliquot of the 24 h time-point added with 0.5 μg of both 4sU labeled and unlabeled spike-in RNAs. RNA-seq libraries were prepared for each time-points and seady-state sample using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) automated on the NGS WorkStation (Agilent) at The Centre for Applied Genomics (TCAG) according to the manufacturer's instructions. PCR cycle numbers were determined using the PCR Add-on Kit for Illumina (Lexogen). All steady-state samples were processed with 250 ng of total RNA input. To minimize variability between time-points within a batch, RNA samples were processed with the same total RNA input with a minimum of 100 ng of total RNA used. Each sample was spiked-in with ERCC RNA Spike-In Control Mix 1 (Ambion) according to the manufacturer's instructions prior to the start of library preparation. Library quality and quantity were measured at TCAG prior to sequencing with Bioanalyzer (Agilent) and KAPA qPCR (Roche). Sequencing was also performed at TCAG on the Illumina HiSeq 2500 with single-end 100bp read length yielding 40 to 50 million reads.