GSM8195878: uninfected PHH, 5C treated, sequencing replicate 2; Homo sapiens; RNA-Seq
Freshly isolated or in vitro cultured hepatocytes (mock or treatment) were homogenized in TRIzol solution and cryopreserved at -80°C. RNA was subsequently isolated using the Zymo research Direct zol RNA purification kit (Cat. No. #R2053) RNA sequencing libraries from isolated/purified RNA were prepared using the Kapa mRNA Hyperprep Kit (Cat. No. #KK8581) using slight modifications of the manufacturer's instructions for mRNA enrichment method. The input total RNA for the various libraries was selected between 150ng, 250ng or 500ng. The enriched mRNA was fragmented at 94°C  for 6min and dA tailing performed at 55°C for 15min. In order to better capture the AT rich Pf transcriptome we modified the PCR protocol as follows: Initial denaturation at 98°C for 2min; Cycling denaturation at 98°C for 20sec, annealing + extension at 62°C for 2min; final extension at 62°C for 3min. We selected PCR cycles for library amplification (12, 11 and 10 respectively) based on starting input total RNA amount.