RNA-Seq of Akkermansia muciniphila MucT
Cell pellets were harvested during mid-log growth and RNA was extracted using the RiboPure Bacteria Extraction Kit. RNA samples were sequenced by Admera Health (South Plainfield, NJ, USA) using the Illumina Sequencing HiSeq platform (Illumina, CA, USA). Quality control of RNA samples was assessed using High Sensitivity RNA ScreenTape on TapeStation (Agilent Technologies Inc., CA, USA) and quantified using the Qubit 2.0 RNA High Sensitivity assay (Thermofisher, MA, USA). Ribosomal RNA depletion was performed with Ribo-Zero Plus rRNA Removal Kit (Illumina Inc., CA, USA). Samples were primed and fragmented based on the recommendation of the manufacturer. First strand synthesis was performed with Protoscript II Reverse Transcriptase using a longer extension step of ~30 min at 42C. NEBNext UltraTM II Non-Directional RNA Library Prep Kit for Illumina (New England BioLabs Inc., MA, USA) was used for the remaining steps of library construction. Final library size was around 350bp with an insert size of 200bp. The final quantity of the library was quantified using the Qubit 2.0 and the quality assessed by the TapeStation D1000 ScreenTape (Agilent Technologies Inc., CA, USA). For the Illumina HiSeq, 8-nt dual-indices were used and equimolar pooling of libraries was done based on the values from the QC before sequencing with a read length configuration of 150 PE for 20 M PE reads per sample (20M in each direction).