RNA-Seq of Streptococcus pneumoniae D39: lacks media with lactose (25mM) at 0.120 OD replicate 2.
The quantity and quality of the purified total RNA was assessed using a Thermo Fisher Scientific Qubit 4.0 fluorometer with the Qubit RNA BR & HS Assay Kits (Thermo Fisher Scientific, Q10211 & Q32855) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (Agilent, DNF-471), respectively. Thereafter, 100 ng of input RNA was depleted of ribosomal RNA using an Illumina Ribo-Zero plus rRNA Depletion Kit (illumina, 20037135). Ribodepleted RNA were made into cDNA libraries using a TruSeq Stranded Total RNA Human/Mouse/Rat Kit (illumina, 20020597) with IDT for Illumina TruSeq RNA UD Indexes (illumina, 20022371). This was done according to the reference guide entitled "TruSeq Stranded Total RNA with Illumina Ribo-Zero Plus rRNA Depletion (illumina, Document # 1000000092426 v01). The resulting cDNA libraries were evaluated using a Thermo Fisher Scientific Qubit 4.0 fluorometer with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32854) and an Agilent Fragment Analyzer (Agilent) with a HS NGS Fragment Kit (Agilent, DNF-474), respectively. The libraries were equimolar pooled and sequenced with a loading concentration of 750 pM, paired-end and dual indexed, using an illumina NextSeq 1000/2000 P2 XLEAP-SBS Reagent kit (illumina, 100 cycles kit, 20100987) on an illumina NextSeq 1000 instrument. The quality of the sequencing run was assessed using illumina Sequencing Analysis Viewer (illumina version 2.4.7) and all base call files were demultiplexed and converted into FASTQ files using illumina bcl2fastq conversion software v2.20. The quality control assessments, rRNA depletion, generation of libraries, sequencing and demultiplexing were carried out at the Next Generation Sequencing Platform, University of Bern.