GSM9196681: 24 hpi, Donor 3, mock; Homo sapiens; RNA-Seq
Total RNA was extracted using Direct-zol RNA miniprep kit (Zymo Research) according to manufacturer's instructions. RNA quality and quantity were analyzed using the NanoDrop One (Thermo Scientific) and Agilent Bioanalyser PHH sequencing library preparation was performed according to NNSR priming method as described previously with the following modifications. One μg of total RNA was used for mRNA isolation using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). An RNA fragmentation step of 5 minutes at 95oC in the buffer of the Maxima H- reverse transcriptase (RT) (Thermo Scientific) was performed before the RT reaction, which was done in the presence of Actinomycin D. After the RT, Thermolabile Exonuclease I (NEB) was used to digest the RT primer. Following RNase H (NEB) treatment the 2nd strand cDNA synthesis was performed with Klenow exo(-) (NEB). After a 1.5-volume magnetic bead purification, 10% of the sample volume was subjected to RT-qPCR to determine the number of amplification cycles required for the barcoding PCRs.