Agilent bioanalyzer rna nano chips

Virulent L. interrogans serovar Manilae strain L495 and perRB (M1474) mutant strains with less than three in vitro passages were used in this study. Four independent biological replicates of exponentially grown L. interrogans WT, perRB (M1474) and double perRAperRB mutant strains were harvested and resuspended in 1 ml TRIzol (ThermoFisher Scientific) and stored at -80°C. Nucleic Acids were extracted with chloroform and precipitated with isopropanol as described earlier [59 (link)]. Contaminating genomic DNA was removed by DNAse treatment using the Turbo DNA-free kit (ThermoFisher Scientific) as described by the manufacturer. The integrity of RNAs (RIN > 8.0) was verified by the Agilent Bioanalyzer RNA NanoChips (Agilent technologies, Wilmington, DE).

Total RNA was extracted from parental and resistant RPMI-8226 and L-363-cells in triplicate using the QIAGEN RNeasy mini kit and included the optional digestion with RNAse-free DNAse I (QIAGEN, Hilden, Germany). RNA quality was analyzed on Agilent Bioanalyzer RNA nano chips (Agilent Technologies, Inc., Santa Clara, CA, USA) and revealed RIN values between 9.5 and 9.9. An amount of 50 ng of total RNA was used to generate NGS libraries using the QuantSeq 3′ mRNA FWD kit (Lexogen, Vienna, Austria), which generates libraries of sequences close to the 3′ end of poly(A) RNAs. Libraries with unique barcodes were pooled, quantitated by qPCR and subjected to single-read sequencing on a NextSeq 500 MidOutput flowcell (Illumina, San Diego, CA, USA) as recommended by the manufacturer. After demultiplexing, reads were subjected to the automated QuantSeq data analysis pipeline provided by Lexogen. In a first step, the pipeline performed trimming, quality control, alignment, and read counting from fastqc input. Thereafter, differentially expressed genes were identified by the differential expression (DE) QunatSeq pipeline (Lexogen, Vienna, Austria).

Agilent Bioanalyzer RNA nano chips (Agilent) were used to evaluate the integrity of total RNA and Qubit RNA kit (Life Technologies) to quantitate RNA in samples. 1 μg of total RNA was used for ScriptSeq™ Complete Gold System (Epicentre) to ribodeplete rRNA and further for RNA-seq library preparation. SPRI beads (Agencourt AMPure XP, Beckman Coulter) were used for purification of RNAseq libraries. The library QC was evaluated on High Sensitivity chips by Agilent Bioanalyzer (Agilent). Paired-end sequencing of RNA-seq libraries was done using Illumina HiSeq technology with a minimum of 60 million 2x100bp paired-end reads per sample.