Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: RNA was purified from cells for each sample using the RNAsnap method [1]. First, cell pellets were resuspended in 500 µl of RNA extraction buffer (18 mM EDTA, 0.025% w/v SDS, 1% v/v 2-mercaptoethanol, 95% v/v formamide) by vortexing. Following incubation at 95°C for 7 min, cell debris was pelleted by centrifugation at 16000 × g for 5 min at room temperature. The resulting supernatant was purified using the Clean & Concentrator kit incorporating the on-column DNase digestion step (Zymo Research). Yields were determined using a Qubit 2.0 fluorometer with the RNA BR assay kit (ThermoFisher). Then, 1.5 µg of purified RNA for each sample was processed using the Ribozero rRNA Removal Kit (Gram-negative bacteria) (Illumina) according to the manufacturer's instructions except that reaction volumes were reduced by 50%. Depleted RNA samples were ethanol precipitated. RNA-Seq library preparation from rRNA-depleted samples began by fragmenting them with the NEBNext Magnesium RNA Fragmentation Module (New England Biolabs) for 110 s followed by ethanol precipitation. Fragmented RNA was treated with 10 U of T4 polynucleotide kinase (NEB) in 20 µl reactions containing 1 mM ATP. After another ethanol precipitation, the NEBNext small RNA Library Prep Set for Illumina (Multiplex Compatible) (New England Biolabs) was used to prepare samples according to recommended protocol, except all reaction volumes were reduced 50% and the SR RT primer concentration was reduced by an additional 50% in the RT primer hybridization step. After performing 15 cycles of PCR, samples were ethanol precipitated, and the DNA concentration was determined via the Qubit HS dsDNA assay kit (Thermofisher). For each sample, 200 ng of DNA was run on a 4% EX E-gel (ThermoFisher). DNA with a size of at least ~200 bases was excised and purified using a gel DNA recovery kit (Zymo Research) with elution in 50 µl of nuclease-free water.