GSM8831216: S_coelicolor_M512lpm_2d biol rep 2; Streptomyces coelicolor; RNA-Seq
RNA was isolated from 5 mL culture volume of Streptomyces coelicolor M512 either expressing the caprazamycin or liposidomycin gene cluster (S. coelicolor M512/cpzLK09 and S. coelicolor M512/lpmLK01) using the RNeasy plus kit (Qiagen, Hilden, Germany). Cells were transferred into lysing matrix B tubes (2 mL) and lysed in a MP FastPrep-24 (all supplied by MP Biomedicals, USA) for two times 15 s at 4000 m/s with a cooling step on ice for 4 min between the two rounds of shaking. RNA was eluted in RNase-free water and measured at the nanodrop 1000 spectrometer (VWR International, USA). Contaminating DNA was digested using the RNase-free DNase kit (Qiagen, Hilden, Germany). The RNA was purified using the RNA clean-up kit (Macherey-Nagel, Düren, Germany) and tested for the absence of DNA by PCR. The total RNA was quantified with a Qubit RNA BR Assay Kit (Thermo Fisher) and RNA integrity was checked by an Agilent 2100 BioAnalyzer with the RNA 6000 Pico kit (Agilent). Library preparation was performed with Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus Microbiome according to the manufacturer's instructions. In brief, 100 ng of total RNA per sample were subjected to rRNA depletion, followed by cDNA library construction, adapter ligation and 15 cycles of barcoding PCR. Obtained libraries were quantified with Qubit 1x DNA HS Assay Kit (Thermo Fisher) and the fragment distribution was checked by an Agilent 2100 BioAnalyzer using the High Sensitivity DNA Kit (Agilent). Libraries were subsequently pooled and sequenced on an Illumina NovaSeq 6000 device using the NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles) with a run mode of 75,10,10,0. The average number of obtained reads was 27-43 million. The sequencing was demultiplexed with the latest version of the nextflow pipeline: nf-core/demultiplex. For demultiplexing bcl2fastq was used and the quality was checked with fastp.