Ribo zero rrna removal kits for gram negative bacteria

Ten to 15 ml of bacteria culture was pelleted by centrifugation at 3,500 g for 10 minutes at room temperature. Two to 4 ml of RNAprotect (Qiagen) was added to the pellet and mixed by vortexing. The pellet was re-precipitated by centrifugation at 3,500 g for 10 minutes at room temperature, and the supernatant was discarded. Standard TRIzol (Life Technologies) protocol was then applied to extract total RNAs from the cell pellets. To ensure that the DNA was completely removed, DNase digestion was performed and the total RNA samples were further purified by acidic phenol–chloroform. mirVana (Life Technologies) was used to deplete tRNAs and other small RNA portions, and the large RNA molecules were retained. Ribosomal RNAs were then removed by Ribo-Zero rRNA removal kits for gram-negative bacteria (Epicentre). External RNA Controls Consortium (ERCC) RNA Spike-in Control Mixes (Ambion) were added to each rRNA-depleted RNA sample according to the user guide. The sequencing libraries were prepared using a ScriptSeq v2 RNA-seq Library Preparation kit (Epicentre) with rRNA-depleted samples, and all of the libraries were sequenced by Illumina HiSeq 2000 following the strand-specific sequencing protocol for 100 cycles. All RNA-seq data have been deposited to SRA with the accession SRP075337.

Ten to 15 ml of bacteria culture was pelleted by centrifugation at 3,500 g for 10 minutes at room temperature. Two to 4 ml of RNAprotect (Qiagen) was added to the pellet and mixed by vortexing. The pellet was re-precipitated by centrifugation at 3,500 g for 10 minutes at room temperature, and the supernatant was discarded. Standard TRIzol (Life Technologies) protocol was then applied to extract total RNAs from the cell pellets. To ensure that the DNA was completely removed, DNase digestion was performed and the total RNA samples were further purified by acidic phenol–chloroform. mirVana (Life Technologies) was used to deplete tRNAs and other small RNA portions, and the large RNA molecules were retained. Ribosomal RNAs were then removed by Ribo-Zero rRNA removal kits for gram-negative bacteria (Epicentre). External RNA Controls Consortium (ERCC) RNA Spike-in Control Mixes (Ambion) were added to each rRNA-depleted RNA sample according to the user guide. The sequencing libraries were prepared using a ScriptSeq v2 RNA-seq Library Preparation kit (Epicentre) with rRNA-depleted samples, and all of the libraries were sequenced by Illumina HiSeq 2000 following the strand-specific sequencing protocol for 100 cycles. All RNA-seq data have been deposited to SRA with the accession SRP075337.