GSM9177446: WT vector, ATc 10, Rep 1; Clostridioides difficile; RNA-Seq
For RNA isolation, samples were pelleted at 4,000 x g for 10 minutes at 4°C, washed with 500 µL TE buffer, and pelleted again. Pellets were resuspended in 1 mL TRIzol (Invitrogen 15596026) and lysed by bead beating twice for 90 seconds, with 90 seconds on ice between rounds. Samples were vortexed upon the addition of 200 µL chloroform, incubated at room temperature for 5 minutes, then centrifuged at 14,000 x g for 10 minutes at 4°C. The aqueous phase was then added to 1 volume of isopropanol, incubated for 10 minutes at room temperature, and centrifuged again at 14,000 x g for 10 minutes. The pellet was washed with 75% ethanol then centrifuged again. The supernatant was removed and the pellet was air-dried for 10 minutes before RNA was suspended in 50 µL nuclease-free water. Purified RNA was submitted to Genewiz (Azenta Life Sciences) for paired-end sequencing using the following pipeline, with kits used per manufacturer's instructions. rRNA was depleted using the Ribo Zero rRNA Removal Kit (Illumina). RNA sequencing libraries were prepared with the NEBNext Ultra RNA library prep kit for Illumina (NEB) and checked using a Qubit 2.0 fluorometer. The libraries were multiplexed for sequencing using a 2 × 150 paired-end configuration on an Illumina HiSeq 2500 instrument. Image analysis and base calling were done using the HiSeq control software. The resulting raw sequence data files (.bcl) were converted to the FASTQ format and demultiplexed with bcl2fastq 2.17 software (Illumina). One mismatch was permitted for index sequence identification.