Ovation human ffperna seq multiplex system

Total RNA was collected for each cell line and treatment condition in three independent replicates. 100 ng of total RNA was used for library preparation using Ovation Human FFPERNA-seq multiplex system (NuGEN Technologies, San Carlos, CA). Sequencing was performed on an Illumina NextSeq500 instrument with 75 bp paired end reads. On average, 23 million pairs of reads were generated for each sample. The average alignment rate was 92.5% with Q30 ≥ 94%.

RNA-Seq libraries for the 26 sputum samples and 4 culture samples were prepared with 200 ng of corresponding RNA using the Ovation Human FFPE RNA-Seq multiplex system (NuGen), which includes proprietary oligonucleotides for the removal of human rRNA and customized oligonucleotides to remove the rRNA of M. tuberculosis. The cDNA was sheared to ∼200 bp with a Covaris E220 ultrasonicator (Covaris) prior to adaptor ligation and amplification. All cDNA libraries were quantified using a Qubit fluorometer and quality checked using a DNA-1000 kit (Agilent) on a 2100 Bioanalyzer. Each sputum library was loaded onto a single lane in a flow cell and sequenced with a Hi-Seq 2500 instrument (Illumina). With the exception of four samples (Rv_E1, Rv_S1, SP55, and SP61) where only ∼100 million 100-bp single-end reads were obtained, all other sputum samples and laboratory cultures (Rv_E2 and Rv_S2) generated ∼200 million 100-bp single-end reads.