title: GSM5049469: Clostridioides difficile monoculture rep3; Clostridioides difficile; RNA-Seq
library_construction_protocol: Samples were stored at -80 °C until used for RNA extraction. Samples were thawed on ice, pelleted, and resuspended in 750 µL of LETS buffer (1 M LiCl, 0.5 M EDTA, 1 M Tris pH7.4). Cells were transferred to tubes containing lysing matrix B beads (MP Biomedicals) and lysed by a FastPrep-24 (MP Biomedicals) bead beater for 45 s at 6 m/s. Lysed samples were heated for 5 min at 55 °C and pelleted by centrifugation for 10 min. The supernatant was transferred to a fresh tube and 1 mL TRIzol (Thermo Scientific) was added. Chloroform (200 µL) was added to each sample and vortexed prior to separation of the aqueous and organic layers by centrifugation for 15 min. The aqueous (upper) layer was transferred to a fresh tube and the RNA was precipitated through the addition of 1 mL isopropyl alcohol. Samples were incubated for 10 min and RNA was pelleted by centrifugation for 10 min. Supernatant was removed and the RNA pellet was washed with 200 µL of 70% ethanol. Samples were air dried for 1 min, then resuspended in 100 µL RNase free water. DNA contamination was removed through the addition of 8 µL RQ1 DNase, 12 µL 10x RQ1 buffer, and 2 µL RNase inhibitor (Promega) to the purified RNA. Samples were DNase treated for 2 h and purified using the RNeasy miniprep RNA cleanup kit (Qiagen). RNA was quantified and either used for RNA sequencing or quantitative PCR (qPCR) as described below. RNA-seq library construction and sequencing were performed by HudsonAlpha Institute for Biotechnology (Huntsville, AL). The concentration and integrity of extracted total RNA were estimated by a Qubit 2.0 Fluorometer (Invitrogen) and an Agilent 2100 Bioanalyzer (Applied Biosystems), and 500 ng of RNA was utilized for downstream applications. rRNA was removed using the Ribo-Zero Gold (Epidemiology) kit (Illumina) according to the manufacturer's instructions. RNA was fragmented and primed for first-strand synthesis using the NEBNext first strand synthesis module (New England BioLabs). Directional second-strand synthesis was performed using the NEBNext Ultra Directional second strand synthesis kit. Libraries were then prepared from samples using the NEBNext DNA Library Prep master mix set (Illumina) with the following slight modifications. End repair was performed and followed by polyadenylic acid (poly[A]) addition and custom adapter ligation. Ligated samples were individually barcoded with unique in-house Genomic Services Lab (GSL) primers and amplified through 12 cycles of PCR. Library quantity was assessed by a Qubit 2.0 Fluorometer, and the library quality was assessed by utilizing a DNA High Sensitivity Chip on a Caliper GX (PerkinElmer). The quantitative PCR (qPCR)-based Kapa Biosystems library quantification kit (Kapa Biosystems) was used for final accurate quantification of libraries prior to sequencing. Each library was diluted to a final concentration of 12.5 nM and pooled equimolar prior to clustering. Paired-end sequencing was performed on an Illumina HiSeq2500 sequencer (Illumina).