GSM9500186: Sample 15; unclassified Streptomyces; RNA-Seq
Streptomyces mycelia was scraped from the agar surface with a spatula, and frozen in liquid nitrogen before storage at -80 °C. Frozen cells were transferred to Lysing Matrix E 2ml tubes (MP Biomedicals LLC) before lysis with a FastPrep bead homogeniser (MP Biomedicals LLC) set to 6.0 m/s for 20 s. Trizol (1ml, ThermoFisher) was added to each tube, mixed by hand inversion and incubated for 5 minutes to allow dissociation of the nucleoproteins complex. Samples were centrifuged at 16,000 x g for 10 min, and 700 µl of supernatant was collected. To each, 140 µl of chloroform was added with incubation at room temperature for 3 min with occasional mixing by hand. After centrifugation for 15 min at 12,000 x g at 4 °C, samples separate to a lower red phenol-chloroform, interphase and colourless upper aqueous phase. The aqueous phase (2 x 140 µl) containing the RNA was transferred to a fresh tube, before removal of 200 µl with the tube tilted to avoid any contamination of the other phases. An equal volume of 200 µl 100% ethanol was added to each. RNA isolation from the resulting samples was performed using the Direct-zol RNA Miniprep kit (Zymo Research) with the manufacturer's protocol, omitting the on-column DNAse treatment (that had proven ineffective in this experiment as indicated by qPCR). Samples were eluted in 50 µl autoclaved milli-Q water and concentration monitored by Qubit HS and BR RNA assay kits (Thermofisher). To remove residual gDNA in 50 µl samples, the manufacturer's protocol was followed for TURBO DNase kit (Ambion), with 1 µl DNase per 50 µl input sample, including the DNase removal steps by kit reagent and centrifugation. RNA integrity was measured by recording RINs with a Bioanalyzer 2100 (Agilent). Library preparation and sequencing was performed by Source Bioscience. The libraries were prepared with the NEBNext rRNA Depletion Kit for Bacteria (NEB) according to the manufacturer's protocol. During this process, the libraries were indexed with NEBNext Multiplex Oligos for Illumina (NEB). Illumina sequencing was performed at depth 30 million read pairs per sample as 150 b.p. paired end reads. RNA samples were multiplexed before library generation and subsequent sequencing. To confirm our assertion that all four strains could be multiplexed without significant data loss, preliminary RNA-seq was performed with samples from strains B and C run individually, compared with the same samples mixed with each other and all four strains. No significant changes were observed in gene expression due to multiplexing, and therefore this strategy was applied to all further RNA-seq.