title: RNA seq of mus musculus: bone marrow
design: Bone marrow-derived macrophages (BMM) from female BALB/c mice were prepared and incubated at 37°C with 5% CO2 atmosphere. BMMs were cultured in cultivation dishes (100 mm x 20 mm) with 10 ml L-cell conditioned medium. After 5 days of incubation, 10 ml fresh medium without antibiotics was added and the cells were incubated further for 5 days. After 10 days of incubation, BMMs were washed three times with PBS and incubated with fresh RPMI 1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (FBS). The cells were then infected with 107 CFU of the virulent strain Brucella abortus for 1 h at 37oC in 5% CO2. Washing was performed using RPMI 1640 medium and then incubated for 24 h with fresh RPMI 1640 medium containing 10% (v/v) FBS and gentamicin (30 µg/ml) to kill extracellular bacteria at 37oC in 5% CO2. Total RNA from the uninfected or infected BMMs were isolated in accordance with the Qiagen RNeasy Mini kit “Animal Cells I” 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: 31358_1