design: Total RNA of C. tardaugens cells was extracted from cultures grown in minimal medium with 20 mM CDX and testosterone as carbon source. Cells where harvested in mid exponential phase (OD600 0.6) and stored at - 80 C. Pellets where thawed and cells were lysed in 400 L TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) containing lysozyme (50 mg/mL) following three freezingthawing cycles. High Pure Isolation Kit (Roche), followed by DNA-free DNA Removal Kit (Invitrogen) treatment, was used to obtained pure RNA. RNA degradation and contamination was monitored on 1% agarose gels.RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA).RNA concentration was measured using Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA).RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).Ribosomal RNA was removed from the total RNA with Ribo-Zero rRNA Removal Kit to later construct a 150 bp paired-end library using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse TranscriptaseRNaseH-. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 l USER Enzyme (NEBUSA) was used with size-selected, adaptor-ligated cDNA at 37C for 15 min followed by 5 min at 95C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.The clustering of the index-coded samples was performed on a cBot Cluster Generation System using HiSeq PE Cluster Kit cBot-HS (Illumina) according to the manufacturers instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.