title: GSM3592340: Phaeobacter inhibens ∆65kb_1B; Phaeobacter inhibens; RNA-Seq
library_construction_protocol: The pestled bacterial cell powder was covered with 1 ml TRIzol reagent (ThermoFisher Scientific, Waltham, MA, USA) and incubated for 5 min at room temperature. The further steps for total RNA isolation starting with the centrifugation at 12,000g for 10 min at 4°C were performed with the RNeasy mini kit (Qiagen, Hilden, Germany) as previously described (Wang et al. 2014). After DNase treatment, the total RNA was resolved in 50 µl ultrapure DEPC-treated H2O. Successful removal of genomic DNA was verified via PCR with specific primers against the 78 kb chromid (P737: 5´-GCTCTGCACCATTTCTTTTCG-3´,  P738: 5´-CGAGGCGGTTTGGTGTGTAGT-3´) using 1 µl of total RNA as a template. Depletion of ribosomal RNA was performed with the mRNA Ribo-Zero Magnetic Kit (Epicentre, Madison, WI, USA) according to the manufacturer´s instructions. Single end, strand specific cDNA libraries were prepared using the Scriptseq v2 RNA-Seq Library Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturers protocol. For sequencing equal volumes of libraries (12 pM) were multiplexed on a single lane. Sequencing was done on the HiSeq 2500 (Illumina, San Diego, CA, USA) using TruSeq SBS Kit v3 - HS (Illumina, San Diego, CA, USA) for 50 cycles resulting in 50 bp reads. Image analysis and base calling were performed using the Illumina pipeline v 1.8 (Illumina, San Diego, CA, USA).
attributes.tag: GEO Accession
attributes.value: GSM3592340