GSM5885551: Spn D39 wt, Rep3; Streptococcus pneumoniae D39; RNA-Seq
Pellets were thawed on ice and resuspended in 1 ml of TRI Reagent (catalog no. TR-118; Molecular Research Center). Resuspended cells were placed in tubes containing 0.1-mm silica beads (Lysing matrix B tubes, catalog no. 6911-100; MP Biomedicals) and lysed in a bead beater (Bead Ruptor 24 Elite; Omni) at a rate of 6 m/s for 45 s twice, with a 5-min cooling on ice between runs. Then, 200 μl of chloroform was added, followed by vigorous mixing and centrifugation for 15 min at 13,200 rpm. The top aqueous layer was removed to a new tube, and 70% ethanol was added at a 1:1 volume ratio. RNA purification was completed using the RNeasy minikit (catalog no. 74104; Qiagen) following DNase I treatment (catalog no. 79254; Qiagen). Integrity of the RNA was checked with Agilent Tapestation, samples with RIN >7 were processed using the following steps. From each sample, 1ug of total RNA was used for ribosome RNA depletion with the Illumina ScriptSeq Bacterial RNA Library.  After rRNA depletion, the libraries were prepared following the rest of the Illumina ScriptSeq Bacterial RNA library preparation kit protocol. The cleaned adapter ligated libraries were pooled and loaded on the NextSeq 500 with a 75 cycle sequencing module to generate paired-end reads. Finally, the demultiplexing of the reads was performed with bcl2fastq version 2.20.0 pipeline