RNA-Seq of intracellular B. melitensis M5-90: infection of macrophages cell RAW264.7
Total RNA from intracellular Brucella was extracted using RiboPure-Bacteria Kit (Invitrogen) with modifications. In RNA purification step the Filter Cartridge (provided with the kit) was omitted, and instead the RNA was precipitated with isopropanol for more than 8h. Genomic DNA was removed using the RQ1 RNase-free DNase (Promega), at 37 for 1h. The isolated RNA was reacquired through Phenol/Chloroform extraction and stored at -80 until use. RNA quality was assessed using the Agilent Bioanalyzer 2100 (Agilent) and spectrophotometry reader (Implen P330). 10 g total RNA samples were used to construct strand-specific dUTP library. The ribosomal RNAs 16s and 23s were removed from purified total RNA by using Ribo-Zero Magnetic kit (G+/G-Bacteria). All RNAs were digest into short fragments. RNA fragments were reverse transcribed to single-stranded cDNAs, the complementary sequence was synthesized by using dUTP. Attach the 5 DNA/DNA adaptor to the cDNAs. Three replicates were included for each treatment and a total of 6 libraries were constructed. The constructed cDNA library was sequenced on an HiSeq 4000 platform (Illumina) and a 150-bp paired-end reads were generated.