title: RNA-seq Human: Brucella2
design: Each sample used a total of 1  g of total RNA as input material for preparing the lncRNA library. According to the manufacturer's recommendation, use NEBNext   UltraTM RNA Library Prep Kit for Illumina  Generate a chain specific library (NEB, USA) and add the index to the attribute sequence of each sample. In short, ribosomal RNA is removed from total RNA in NEBNext First Strand Synthesis In the Reaction Buffer (5X), divalent cations are used for fragmentation at high temperatures. Synthesize first stranded cDNA using random hexamer primers and M-MuLV reverse transcriptase (RNase H -). Subsequently, DNA polymerase I and RNase H were used for second stranded cDNA synthesis, where dUTP was incorporated instead of dTTP. The remaining protruding ends are converted into flat ends through exonuclease/polymerase activity. After adenosylation at the 3 'end of the DNA fragment, it is hybridized by connecting to a NEBNext adapter with a hairpin ring structure. In order to select cDNA fragments with a length priority of 250-300 bp, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Then, the second strand was digested at 37  C using a size selected, spliced cDNA using USER Enzyme (NEB, USA) for 15 minutes, followed by digestion at 95  C for 5 minutes, followed by PCR. Then PCR was performed using Phusion high fidelity DNA polymerase, universal PCR primers, and index (X) primers. After the construction of the library is completed, initial quantification is performed using Qubit2.0 Fluorometer to dilute the library to 1.5ng/ul. Subsequently, the insert size of the library is detected using Agilent 2100 bioanalyzer After meeting the expected size, qRT PCR accurately quantified the effective concentration of the library (the effective concentration of the library is higher than 3nM) to ensure the quality of the library.