GSM8140827: C. senegalense IStron-2 total RNA-seq, heterologous; Escherichia coli; RNA-Seq
RNA from E. coli was extracted by resuspending cell pellet in 1 ml Trizol and incubating at room temperature for 10 min before adding 200 ul chloroform. The tubes were mixed and centrifuged at 12,000 g for 15 min at 4 °C. The aqueous phase was transferred to a new tube and purified using Monarch RNA Cleanup Kit (NEB). RNA was stored at -80 °C until library preparation. RNA from C. senegalense cell pellets were extracted in 96-well format using a silica bead beating-based protocol adapted from a prior study Ji et al., 2019, Nat Methods. Briefly, 200 µL of 0.1 mm zirconia silica beads (Biospec,) were added to each well of 96-well deep-well plates (Thermo Fisher Scientific). Next, cell pellets were resuspended in 500 µL DNA/RNA shield buffer (Zymo Research) and transferred to separate wells, and the plates were affixed with a sealing mat and centrifuged for 1 min at 4,500 g. To avoid overheating, the plates were vortexed for 5 s and incubated at −20 °C for 10 min before beating. Then, plates were fixed on a bead beater (Biospec) and subjected to bead beating for 5 min, followed by a 10 min cooling period. The bead beating cycle was repeated three times, and plates were then centrifuged at 4,500 g for 5 min to remove cell debris. Next, 60% of the bead beating volume was transferred to the Quick-RNA Miniprep Plus kit (Zymo Research), and RNA was purified using the manufacturer's protocol for gram positive bacteria. RNA quality was assessed using the 260/280 nm ratio (~2.0) as measured by Nanodrop, and concentration was measured by the Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific) using the manufacturer's protocol. RNA was stored at -80 °C until library preparation. For total RNA-seq library preparation of samples from E. coli, 200 ng of purified total RNA was depleted for ribosomal RNA with Ribo-Zero Plus rRNA Depletion kit (illumina) following the manufacturers protocol and used for downstream library preparation. For samples from C. senegalense, 10 µg of purified RNA was treated with Turbo DNase I (Thermo Fisher Scientific) for 1 h at 37 °C using the manufacturer's protocol. A 2× volume of Mag-Bind TotalPure NGS magnetic beads (Omega) was added to each sample, and the RNA was purified using the manufacturer's protocol. The RNA was then diluted in NEBuffer2 (NEB) and fragmented by incubating at 92 °C for 1.5 min. To generate RNA with 5′-monophosphate and 3′-hydroxyl ends, samples were treated with RppH (NEB) supplemented with SUPERase•In RNase Inhibitor (Thermo Fisher Scientific) for 30 min at 37 °C, followed by T4 PNK (NEB) in 1× T4 DNA ligase buffer (NEB) for 30 min at 37 °C. Samples were column-purified using RNA Clean & Concentrator-5 (Zymo Research), and the concentration was determined using the DeNovix RNA Assay (DeNovix). Illumina adapter ligation and cDNA synthesis were performed using the NEBNext Small RNA Library Prep kit. Dual index barcodes were added by PCR amplification (12 cycles), and the cDNA libraries were purified using the Monarch PCR & DNA Cleanup Kit (NEB). High-throughput sequencing was performed on an Illumina NextSeq 550 in paired-end mode with 75 cycles per end for E. coli samples and 150 cycles per end for C. senegalense samples.