GSM3103087: Staphylococcus epidermidis Ctr and α-MG; Staphylococcus epidermidis; RNA-Seq
After incubation, 2 volume of RNAprotect Bacteria Reagent (Qiagen) was added to one volume of bacterial cultures. Then, the cells were pelleted by centrifugation, resuspended in 200 µl of TE buffer containing 15 mg/ml of lysozyme and 10 µl of proteinase K and incubated at 37°C for 30 min. After incubation, zirconia beads (50 mg; 0.1 mm) were added to lyse the cells using FastPrep 24 instrument (Qbiogene) and the total RNA was extracted using RNeasy Mini kit (Qiagen). To remove genomic DNA, on-column DNA digestion was done using RNase-Free DNase set (Qiagen). The RNA integrity was analyzed using LabChip GXII Touch HT (PerkinElmer). The ribosomal RNA (rRNA) was depleted using Ribo-Zero rRNA Removal kit for Gram-positive bacteria (Illumina). Sequencing library was prepared as described before (Macosko et al., 2015) with modifications. Reverse transcription of mRNA was performed using specially designed primers. Similarly to the method described in (Macosko et al., 2015), all the primers shared a common sequence that served as a PCR handle. Additionally, the primers contained known 12 bp barcode (unique for each sample) and different random unique molecular identifiers (UMIs) (Macosco et al., 2015). The poly(T) regions of the oligos were replaced by random hexamers to enable the capturing of the bacterial mRNA particles. The reverse transcription, PCR amplification of the cDNA and tagmentation were performed as described in (Macosko et al., 2015). Paired-end sequencing was performed on NextSeq500 sequencer (Illumina) using the sequencing primers described in (Macosko et al., 2015) in Biomedicum Functional Genomics Unit (FuGU; Helsinki)