Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: RNA sequencing was performed on the wild type strain L. plantarum NZ3400, the auto-aggregating strains IA01t0 and PA4_02 and the non-aggregating IA01t4. Pre-cultures were performed in MRS broth at 30°C overnight. Experiments were performed in triplicates for NZ3400, IA01t0 and IA01t4 and in duplicate for and PA4_02. Cultures were inoculated from an overnight pre-culture and grown until OD600nm=2.6-2.7. The growth phase was validated via pH measurements and HPLC analysis of glucose and lactate concentrations. Total RNA was extracted based on a chloroform/phenol extraction followed by purification using the High Pure RNA isolation kit (Roche Diagnostics, Rotkreuz, Switzerland) as described previously (58). RNA quantity, purity and integrity were verified using an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Samples with an RNA integrity number (RIN) > 9 and a 16S/23S-rRNA ratio > 1.5 were selected for rRNA depletion. Thereafter, EDTA was added to 1 mM and depletion was performed using the MICROBExpress™ Bacterial mRNA Enrichment Kit (Life Technologies Europe BV, Zug, Switzerland) following the manufacturer's instructions. Concentrations of depleted samples were determined in a TapeStation and normalized to 100 ng/ml using in Tris-HCl (10 mM, pH=8.5). Sequencing of 100 bp single reads was done on Illumina Novaseq 6000 (Illumina Inc., California, USA) at the Functional Genomics Center Zurich (FGCZ). The library was prepared according to the Illumina Truseq Total RNA protocol.