RNA-Seq of Caldimonas thermodepolymerans DSM 15344: TBS cultivation replicate 1
Cultures were pelleted and stabilized using RNA protect cell reagent (Qiagen), and then the total RNA was extracted from these cell pellets using Promega SV Total RNA Isolation System according to the manufacturers instructions with few modifications. An additional lysis step was added to the protocol as an initial step using a freshly prepared Tris-EDTA buffer (10 mM Tris base and 1 mM EDTA-Na2HO. pH 7.5) containing lysozyme. DNase treatment was done firstly by performing an on-column treatment was done according to the manufacturers procedures. After finishing the RNA extraction procedure, an additional treatment was done using TURBO DNA-free kit (Thermo Fisher). A rigorous DNase treatment was applied to the undiluted samples to remove all DNA contaminants from the RNA samples according to the manufacturers protocol. RNA samples were stored at -80C till further processing. The total RNA quantity was determined with the Quant-it Ribogreen RNA assay (Thermo Fisher Scientific) and RNA integrity was evaluated on a Bioanalyzer instrument with an RNA 6000 Nano chip (Agilent Technologies). Ribosomal RNA depletion of 550 ng of total RNA was established using a PAN-Bacteria riboPOOL kit (siTOOLS), followed by purification with a Zymo RNA Clean and Concentrator-5 kit (Zymo Research). Sequencing libraries were subsequently prepared in NxtGnt with the Xgen stranded RNA library prep kit for Illumina (Integrated DNA Technologies) in combination with the Xgen UDI 8nt primer pairs for Illumina (Integrated DNA Technologies). Library enrichment PCR proceeded for 14 cycles and the libraries were purified with 0.8x AMPure XP beads (Beckman Coulter). Additional purification of the libraries on an Egel EX 2% (Thermo Fisher Scientific) was performed to get rid of left-over adapter dimers. Library quality was verified on a Bioanalyzer instrument with a DNA High Sensitivity chip (Agilent Technologies) and concentrations were measured with qPCR according to the Sequencing library quantification guide (Illumina). Sequencing was performed on a NextSeq500 instrument (Illumina) in high output with single reads of 75 nts and 3% Phix spike-in.