Sequencing library qpcr quantification protocol guide

For each condition, three biological replicates were sequenced. RNA concentrations were measured with the Quant-iT RiboGreen RNA assay (Life Technologies, Inc.), and RNA quality was assessed by capillary electrophoresis with the RNA 6000 Pico Chip (Agilent Technologies). A 2-µg sample of RNA was depleted of rRNA with the Ribo-Zero rRNA Removal kit for Gram-negative bacteria (Illumina). Library preparation was performed in accordance with the TruSeq Stranded Total RNA Library Preparation protocol (Illumina). Libraries were quantified by qPCR in accordance with the Illumina Sequencing Library qPCR Quantification protocol guide, version February 2011. Library size distribution and quality were checked on a DNA 1000 chip (Agilent Technologies). Sequencing was performed with a high-throughput Illumina NextSeq 500 flow cell generating 75-bp single reads.
Reads were mapped to the B. cenocepacia LMG 16656 reference genome (6 (link)) with a 95% similarity cutoff by using CLC Genomics Workbench version 8.5.1 (Qiagen). Statistical analyses were performed with EDGE (Estimated Degree of Gene Expression). Genes were reported as significantly differentially expressed when the P value was <0.05 and there was a change of ≥1.5-fold.

The concentration of the
S. gregaria high molecular weight DNA sample was measured with PicoGreen (Invitrogen) fluorimetry, after which DNA integrity was confirmed by gel electrophoresis (1% E-Gel; Invitrogen). The sample was divided for Illumina MP and PE sequencing library preparation.
The MP sequencing library was prepared from 1 µg of the sample with a “Nextera Mate Pair Library prep kit” (Illumina). The PE library was prepared with a “NEBNext Ultra II library prep kit” (NEB) from 2 µg of the sample, sheared to 500 bp fragments using an S2 focused-ultrasonicator (Covaris). Size selection (600–700 bp) was performed for both libraries in a 2% E-Gel (Invitrogen). The quality of the libraries was confirmed with a Bioanalyzer High Sensitivity DNA Kit (Agilent). The MP and PE libraries were quantified by qPCR, according to Illumina's “Sequencing Library qPCR Quantification protocol guide” (version February 2011) and pooled at a molar ratio of 25% MP – 75% PE for sequencing on Hiseq3000 (2 × 150 cycles, 16 lanes; Illumina).