RNA-Seq of U2OS human osteosarcoma cells infected with mycoplasma
Following total RNA extraction using the Qiagen RNeasy mini kit, RNA sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA library prep gold kit following the manufacturers instructions and 150 ng of starting material as described in Taylor et al9. Briefly, 150 ng of total RNA was depleted of ribosomal and mitochondrial ribosomal RNA, cleaned up using RNA clean XP beads (Beckman Coulter, High Wycombe, United Kingdom) and then fragmented. Next, first and second stand cDNA synthesis was conducted, and the resultant cDNA purified using AMPure XP beads (Beckman Coulter) and adenylated at the 3 end. Illumina indexing adapters were then ligated to the cDNA, the fragments purified and then enriched by PCR. Following amplification, the libraries were purified and then their concentration determined using the KAPA Library Quantification Kit for Illumina Platforms following the manufacturers instructions. The libraries were then diluted to 4 nM and pooled in equal volumes prior to sequencing. Paired end RNA sequencing was then conducted using the NextSeq 550 and a Nextseq 500/500 v2 75 cycle kit, with the library loaded at 1.8 pM. Trimmed reads (adapter sequences and low quality ends removed) are provided.