title: GSM8422143: dRNA Seq  ∆modT 9 hours, biol rep 3; Clostridioides difficile; RNA-Seq
library_construction_protocol: RNA was extracted using the Hot Phenol method: Samples were thawed on ice, centrifuged for 20 min, 4500 rpm at 4 °C and the supernatant was completely discarded. Cells were suspended in 600 μl of 10 mg/ml lysozyme in TE buffer (pH 8.0) and incubated at 37 °C for 10 min. Next, 60 μl of 10 % w/v SDS was added and mixed by inversion. Samples were heated in a water bath at 64 °C, 1-2 min before adding 66 μl 3 M NaOAc, pH 5.2. Next, 750 μl acid phenol was added and phases were mixed by shaking followed by an incubation step at 64 °C  for 6 minutes with intermediate mixing. Samples were briefly placed on ice to cool before centrifugation for 15 min, 13,000 rpm at 4 °C. The aqueous layer was transferred into a 2 ml phase lock gel tube (Eppendorf), 750 μl chloroform (Roth, #Y015.2) was added, and everything centrifuged for 12 min, 13,000 rpm at room temperature. The aqueous layer was transferred to a fresh tube, 2 volumes of 30:1 mix (EtOH:3 M NaOAc, pH 6.5) was added and incubated overnight at -20 °C. Precipitated RNA was harvested by centrifugation, washed with cold 75 % v/v ethanol twice and air-dried. Purified RNA was resuspended in 50 µl RNase-free water and stored at -80 °C. From each sample 5 µg RNA were treated with 5U DNaseI (Thermo Fisher) according to the manufacturer's instructions, with 1U/µl RNase Inihibitor (Takara) in a volume of 50 µl and subsequently digested at 37°C for 30 minutes. The sample was adjusted to 100 µl with RNase free water and aliquoted to a phase lock gel tube, along with 100 µl P:C:I for RNA extraction. Phases were mixed by shaking and separated again by centrifugation at 16000 x g, 4 °C for 15 minutes. The aqueous phase was transferred to a new tube, 2.5 volumes of 30:1 Ethanol to NaOAc (3M pH 6.5) and samples were stored at -20 °C overnight for precipitation.  RNA was collected by centrifugation for 30 minutes at 16000 x g, 4 °C, washed twice with 200 µl 75% EtOH and 10 minutes centrifugation steps at 16000 x g, 4 °C. Finally, RNA pellets were air dried for 5 minutes at room temperature, dissolved in 20 µl RNase-free water and stored at -80°C until further processing. Library preparation was performed by vertis Biotechnologie AG. First, ribosomal RNA was depleted using an in-house protocol. Subsequently, RNA was fragmentated by ultrasound (1 pulse of 30 s at 4°C) and an oligonucleotide adapter was ligated to the 3' end. First-strand cDNA synthesis was performed with the M-MLV reverse transcriptase using the appended 3' adapter as primer. The first strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/µl using a high fidelity DNA polymerase with 15 PCR cycles. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). For Illumina NextSeq sequencing, the samples were pooled in approx. equimolar amounts and the cDNA pool was size fractionated in the range of 200 – 600 bp using a preparative agarose gel.