title: Comparison of RNA-seq of Streptococcus suis wild-type and ΔmnmE mutant strains
design: Comparison of RNA-seq of Streptococcus suis wild-type and ΔmnmE mutant strains
library_construction_protocol: The cells were collected at 4°C, and washed twice with precooled PBS buffer, and the pellets were frozen in liquid nitrogen.  All bacteria were cultured at 37°C. 5%CO2 The RNeasy kit (Qiagen) and Ribo-Zero™ rRNA Removal Kit (EPICENTRE Biotechnologies) was used to isolate RNA and remove rRNA. A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the 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 for eucaryote ( For prokaryote, rRNA was removed from total RNA using Ribo-Zero rRNA Removal Kit to purify mRNA). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. First strand cDNA was synthesized using random hexamer primer and RNaseH. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. The library fragments were purified and resolved with EB buffer, then terminal repair、A-tailing and adapter added were implemented. After size selecting and retrieving by AMPure XP beads, the products were used as the index PCR templates. RNA concentration of library was measured using Qubit® RNA Assay Kit in Qubit® 3.0 to preliminary quantify and then dilute to 1ng/μl. Insert size was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA), and qualified insert size was accurate quantification using StepOnePlus™ Real-Time PCR System(Thermo Fisher Scientific，MA，USA)(Library valid concentration＞10 nM).