After RiboGreen quantification and quality control by Agilent BioAnalyzer, 500 ng of total RNA underwent ribosomal depletion with the NEBNext rRNA Depletion Kit (Bacteria) (NEB catalog # E7850) and library preparation with the TruSeq Stranded Total RNA LT Kit (Illumina catalog # RS-122-1202) according to instructions provided by the manufacturer with 8 cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina). On average, 27 million paired reads were generated per sample. Post-run demultiplexing and adapter removal were performed and fastq files were inspected using fastqc (51 ). Trimmed fastq files were then aligned to the reference genome (M. tuberculosis H37Rv; NC_000962.3) using bwa mem (52 (link)). Bam files were sorted and merged using samtools (53 (link)) and gene counts were obtained using featureCounts from the Bioconductor Rsubread package (54 (link)). Differentially expressed genes were identified using the DESeq2 R package (55 (link)) and subsequent analysis of gene expression was performed in (56 , 57 ).
Nebnext rrna depletion kit bacteria
Manufactured by New England Biolabs
The NEBNext rRNA Depletion Kit (Bacteria) is a laboratory tool designed to remove ribosomal RNA (rRNA) from bacterial RNA samples. It is used to enrich for messenger RNA (mRNA) and other non-ribosomal RNA species, enabling more efficient downstream analysis, such as RNA sequencing.
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Lab products found in correlation
Novaseq 6000, by Illumina (1 mentions)
Novaseq 6000 s4 reagent kit 200 cycles, by Illumina (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Truseq stranded total rna lt kit, by Illumina (1 mentions)
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (1 mentions)
Monarch total rna miniprep kit, by New England Biolabs (1 mentions)
Bioanalyzer system, by Agilent Technologies (1 mentions)
Qubit fluorometer, by Promega (1 mentions)
2 protocols using nebnext rrna depletion kit bacteria
1
RNA-Seq Protocol for M. tuberculosis
2
Transcriptome Analysis of lsr2 Mutant Strain
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Wild-type and lsr2 mutant strains were grown until exponential growth phase with optical density value 600 nm (OD600) of 0.6 corresponding to approximately 2.4 × 108 cells/mL. A total of 4.8 × 109 cells were harvested, and total RNA from three biological replicates was extracted for each strain using the Monarch Total RNA Miniprep Kit (NEB). After DNase I treatment and elution, RNA concentrations were measured using the Qubit fluorometer (Promega). RNA integrity was evaluated using the Bioanalyzer system (Agilent), and all samples used for library preparation showed RNA integrity number scores >8. rRNA depletion was performed using the NEBNext rRNA Depletion Kit (Bacteria) (NEB), and the sequencing libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit (NEB) following manufacturer’s instructions. RNA from lsr2 complemented strains used for RT-qPCR experiments was extracted using the Monarch Total RNA Miniprep Kit (NEB).
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