Dual RNA-Seq of Homo sapiens infected with Staphylococcus aureus: infected aortic valve
Ribosomal RNA molecules (human plus bacteria) were depleted using a mixture of siPOOLs human and PANProkaryote rRNA removal solutions (siTOOLs Biotech) or an in-house developed protocol. The ribodepleted RNA samples were first fragmented using ultrasound (1 or 4 pulses of 30 s each at 4C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 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/l using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool was size fractionated in the size range of 200  500 bp / 200  600 bp using a preparative agarose gel. An aliquot of the size fractionated pool was analyzed by capillary electrophoresis. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The following adapter sequences flank the cDNA inserts: TruSeq_Sense_primer i5 Barcode 5'-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT-3 TruSeq_Antisense_primer i7 Index 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 1~75 bp read length.