title: GSM7254813: ZIKV-infected human microglial cells, 16 h, replicate 1, RNAseq [Z16_1]; Homo sapiens; RNA-Seq
library_construction_protocol: Cells (5×104/well) were seeded in 24-well-plates at 37°C and cultured overnight. Cells were pre-treated with metformin at specified concentration (5 mM) for 2 h, followed by ZIKV infection at MOI 3. After infection of 1.5 h, cells were washed three times with PBS. Medium containing metformin was again supplemented and maintained during the culture. The total RNA of samples at 16 and 48 h were extracted for RNA-seq analysis. Preparation of RNA library and transcriptome sequencing were conducted by Novogene (Sacramento, CA). The numbers of reads mapped to each gene were counted using HTSeq v0.6.1. The expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) of each gene was then calculated based on the length of the gene and the read count mapped to this gene. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis  Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp  in length, the library fragments were purified with AMPure XP system (Beckman Coulter,Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptorligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index(X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.