RNA-Seq of Burkholderia cenocepacia
Burkholderia cenocepacia H111 and derived mutant strains were grown in Lenox LB medium (LLB, 1%) tryptone, 0.5% yeast extract, 0.5% NaCl) or M9 medium (0.5% glucose, 0.05% NaCl, 0.6% Na 2 HPO 4 , 0.3% KH 2 PO 4 , 0.1% NH 4 Cl, 1 mM MgSO4 , 0.1 mM CaCl 2 ) at 22-25 or 37C. E. coli strains CC118 pir, SM10 pir and DH5 were grown in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) at 37C. When appropriated, the medium was supplemented with 0.5% glucose, 0.5% rhamnose, kanamycin (50 g/mL), trimethoprim (50 g/mL), gentamicin (20 g/mL), chloramphenicol (30 g/mL), irgasan (25 g/mL) and 1.5% agar (for solid media). For motility assays, semisolid medium (1% tryptone, 0.5% NaCl, 0.5% agar) was used. Total RNA extraction was performed using Direct-zol RNA extraction kit (Zymo Research), from all lbiological replicates. Libraries were constructed with 100 ng total RNA using the Illumina Stranded Total RNA Prep and ligation with Ribo-Zero Plus kit for removed the rRNAs, and quantified by qPCR using Library Quantification Kit (Roche). For strand spacific RNA-seq, the NextSeq 2000 (Illumina) was used. Sequencing was perfomed using 2x100 bp paired-end reads. The sequences in oder to trim the adapters from fastqd files:Adapter Read 1, CTGTCTCTTATACACATCT; Adapter Read 2, CTGTCTCTTATACACATCT.