title: GSM8422129: DMS-MaP, wild type ModT long, deproteinated cell lysate 9h, control; Clostridioides difficile; OTHER
library_construction_protocol: RNA was extracted according to the manufacturer's instuctions. Briefly, 0.2 volumes of chloroform was added, followed by thorough mixing. Samples were centrifuged at 12000 x g for 15 minutes, 4 °C and the aqueous phase was transferred to a new tube in which the RNA was precipitated by adding 1 volume of isopropanol and 1 µl of Glycoblue (Invitrogen) followed by a 30 minutes incubation at -80°C. RNA was centrifuged at 12000 x g, 4 °C for 30 minutes and the pellets were washed twice with 1 ml of 75% Ethanol, air dried, and eluted in 15 µl water. For cDNA synthesis half the volume of RNA (7.5 µl) was heated to 65 °C together with 1.5 µl 100 µM primer (LongModT_Rv for the long ModT isoform, and ShortModT_Rv for short ModT isoform) for 5 minutes and used for reverse transcription with the Marathon-RT (Zhao et al., 2018) in RT-buffer (50 mM Tris–HCl pH 8.3, 200 mM KCl, 5 mM DTT, 20% glycerol, 1 mM MnCl2, 0.5 mM dNTP and 8 U RNaseIn) for 8 hours at 42 °C.  cDNAs were diluted to 1/8 with nuclease-free water. 5 µl the diluted cDNA was used as template in 50 µl PCR reaction using the Q5 High-Fidelity DNA Polymerase (NEB) and the primers HF_ModT_Fw (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGATATTTAGGTCTGTG) and HR_LongModT_Rv (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATATAAAAAAGAGCTATAC) for the long ModT isoform, and HF_ModT_Fw and HR_ShortModT_Rv (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATATTTAGCTGACCTGG)  the  short ModT isoform, appending Nextera sequencing adapter sequences HF and HR to the amplicons extremities. Cycling conditions were 2 min at 98°C then 25 cycles of 10 s at 98°C, 15 s at 55°C and 15 s at 72°C then 72°C for 5 min. 5 µl of PCR products were verified on a 1.5% agarose gel and the rest was column purified using NucleoSpin Gel and PCR Clean-up kits, Macherey-Nagel according to the manufacturer's instructions. A final indexing PCR was carried out using Illumina Nextera DNA CD indexes (96 Indexes, 96 Samples, Illumina). Reaction conditions were 80 ng of purified PCR product, 1 × Q5 reaction buffer, 0.2 mM dNTPs, 2.5 µl of indexing primer, 0.02 U/µl of Q5 polymerase in a final volume of 14 µl. Indexed PCR products were verified on a 1.5% agarose gel, pooled together in an equimolar ratio, before final purification on a 1.5% agarose gel. The pooled indexed sequencing library was quantified using the NEBNext library Quant Kit for Illumina and paired-end PE150 sequencing was carried out on an Illumina Novaseq instrument (Novogene).