title: GSM7310628: Glu3; Clostridioides difficile R20291; RNA-Seq
library_construction_protocol: The cell suspension was thawed on ice, centrifuged at 5 000 x g for 10 min at 4oC, supernatant decanted and the pellet resuspended in 5 mL 1% beta-mercaptoethanol. The suspension was then centrifuged once more at 10 000 x g for 5 min at 4oC. RNA isolation was carried out using a Qiagen RNeasy Mini Kit (Qiagen, Manchester, UK ). Cells were resuspended in 200 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0) containing 15 mg/mL lysozyme and 1.5 mg/ml Proteinase K (Qiagen, Manchester, UK) and incubated for 10 min at room temperature followed by the addition of 700 µL buffer RLT with 1% beta-mercaptoethanol. Cells were then transferred to a 2 mL Lysing Matrix B tube (MP Biomedicals, Cambridge, UK) and treated in a FastPrep-24 instrument (MP Biomedicals, Cambridge, UK) at a setting of 6.0 m/s for 45 s. The lysate was then centrifuged at 13 000 x g for 10 min and the supernatant added to 500 µL of 98% ethanol before RNA isolation and clean-up using a RNeasy column by following the manufacturer's instructions. The protocol was modified to include an initial on-column DNA digestion step using the RNase-free DNase set (Qiagen, Manchester, UK) according to the manufacturer's instructions. RNA integrity was assessed using the Agilent 4200 TapeStation and the Agilent RNA ScreenTape Assay (Agilent, SantaClara, USA) with RNA samples of RIN > 8.0 being used for subsequent procedures. Ribodepletion was performed on 500 ng of total RNA, using the Qiagen FastSelect-5S/16S/23S ribodepletion kit (Qiagen, Manchester, UK) using the protocol: FastSelect-5S/16S/23S with NEBNext Ultra II Directional Library Prep Kit. Indexed sequencing libraries were then prepared using the NEBNext Ultra II Directional RNA Library Preparation Kit for Illumina (New England BioLabs, Ipswich, UK) and NEBNextMultiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (New England Bio Labs, Ipswich, UK). Libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (Thermo Fisher Scientific, Massachusetts, USA). Library fragment-length distributions were analysed using the Agilent TapeStation 4200 and the Agilent High Sensitivity D1000 ScreenTape Assay (Agilent, Santa Clara, USA). Libraries were pooled in equimolar amounts and final library quantification performed using the KAPA Library Quantification Kit for Illumina (Roche; Diagnostics, UK). The library pool was sequenced on the Illumina NextSeq 500 on a NextSeq500 High Output 300 cycle kit (Illumina, San Diego, USA), to generate over 10 million pairs of 150-bp paired-end reads per sample.