title: Illumina HiSeq 1500 sequencing; RNA-seq of murine spleen B-cells after pentanoate treatment compared to untreated control
design: RNA-seq of murine spleen B-cells after pentanoate treatment compared to untreated control
library_name: murine_B_cells_CpG_treated-3_s
library_construction_protocol: The spleen was isolated from FIR/TIGER mice on C57/Bl6 Background (IL-10 (GFP) - Foxp3 (RFP) - reporter mouse). Cells were homogenized with 30 µM cell strainers, B220 positive B cells were isolated using Magnet-associated cell sorting (negative selection) to a purity of over 90% Fresh B cells were either used for RNA isolation directly (murine_B_cells_fresh_control) or treated with 5 µg/ml CpG 2395 for culturing. Cells were cultured in RPMI complete (500000 cells/ well in 500 µL) conditioned with CpG 2395 (5 µg/ml). 24 h after stimulation with CpG 2395 the cells were incubated in presence (murine_B_cells_CpG_and_Pentanoate_treated) or absence (murine_B_cells_CpG_treated) of 5 mM pentanoate (valeric acid) for 4 days at 37°C, 5% CO2 saturation and 95% humidity. Cells were harvested and washed in PBS, RNA-isolation was performed according to the protocol of the Qiagen RNeasy Mini Kit (Qiagen) incl. DNase treatment RNASeq libraries were prepared using a TruSeq Stranded mRNA Sample Prep kit (Illumina, Tokyo, Japan).
attributes.tag: Experimental Factor: compound
attributes.value: CpG2395
attributes.tag: Experimental Factor: dose
attributes.value: 5 microgram per microliter