RNA-seq of Erinaceus roumanicus: oral metagenomics sample
RNA was extracted from rectal and oropharyngeal swabs, using QIAamp viral RNA mini kit (Qiagen, German). 140L of the rectal/oropharyngeal swab sample was added to the mix of RNA carrier and AVL lysis buffer and then incubated for 10 minutes. Next steps were made according to the protocol. RNA was eluted in 60L of the AVE buffer. 10 L RNA was used in next steps, while the rest of RNA was stored at -70. RNA samples have been cleaned of DNA with NEB DNase I (RNase-free). The concentration of total RNA was measured using Qubit RNA HS Assay. First strand of cDNA was obtained using NEBNext Ultra II RNA First Strand Synthesis Module (NEB, England), second strand of cDNA was obtained using NEBNext  Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB, England). End preparation was performed with NEBNext End Repair Module which is a part of NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, Illumina). MGIEasy DNA Adapters (MGI, China) in amount of 1,25 ul were ligated to double stranded cDNA using a ligation module which is also a part of NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, Illumina). Then libraries were amplified with PCR Primer Mix and PCR Enzyme Mix from MGIEasy FS DNA Library Prep Kit. High throughput sequencing was performed using DNBSEQ-G400RS High-throughput Rapid Sequencing Kit (FCS PE100 and FCL PE150) and DNBSEQ-G400RS Rapid Sequencing Flow Cell.