title: RNA seq of brucella abortus: 544
design: Brucella abortus were isolated as follows. BMMs were washed three times with PBS, lysed using sterile distilled water and incubated at 37oC for 5 min. Adherent lysed macrophages were scraped, pelleted at 14,000 x g for 2 min, and the supernatant was removed. To enzymatically digest the macrophage DNA, the pellet was resuspended in a 1 ml solution containing 890 µl sterile water, 100 µl RQ1 DNase Reaction Buffer (Promega), 10 µl RQ1 RNase –free DNase 1 u/µl (Promega). The solution was incubated for 30 min at 37oC and then centrifuged at 8,000 x g for 2 min. The supernatant was removed and the bacteria were pelleted at the bottom. Brucella abortus were washed with PBS and then incubated in 500 µl of RNA-free water for 30 min at room temperature. One milliliter RNA protect Bacteria Reagent (Qiagen) was added directly to sample and incubated for 5 min at room temperature. After centrifugation at 5,000 x g, bacteria were dissolved in 1 ml total solution containing 850 µl RNase-free water (Qiagen), either 150 µl 1% sodium dodecyl sulfate (SDS) or 10% SDS, and 10 µl proteinase K (Promega). The sample was then incubated at 37oC for 1 h, followed by adding 200 µl Chloroform (Sigma). The total RNA was isolated using Qiagen RNeasy Mini “Bacterial” protocol (Qiagen). DNA was removed by using the “On-Column DNase Digestion” protocol. The Ribo‐Zero rRNA removal kit (Epicentre, USA) was used for ribosomal RNA depletion according to manufacturer’s instructions. Libraries for Illumina sequencing were made with the TruSeq Stranded mRNA sample prep kit (Illumina, USA) following the manufacturer’s protocol. RNA sequencing was performed on the Illumina HiSeq 2500 platform using single‐end 50 bp sequencing.
library_name: 31355