title: GSM8974816: Mouse BMDCs treated with 4A-MPLA #1 for 24 h; Mus musculus; RNA-Seq
library_construction_protocol: DCs treated with lipid A analogs were harvested and centrifuged for 1 min at 18,000 rpm at 4°C. After discarding supernatant, cell pellets were treated with 500 μL of TRIzol (Invitrogen) and incubated for 5 min at RT, then for 1 h or overnight at -80°C. Thawed samples were treated with 100 μL of chloroform and then vigorously vortexed for 15 sec. After incubation at RT for 5 min, the samples were centrifuged 15 min at 18,000 rpm at 4°C, and the aqueous fractions were collected into new tubes in ice. 1.0 μL of Glycoblue Coprecipitant (Invitrogen) was treated to each tube, and 250 μL of isopropanol was treated to each sample. After incubation for 1 h at -80°C, the samples were centrifuged for 20 min at 18,000 rpm at 4°C. Supernatants were discarded, and the pellets were washed with 500 μL of 75% ice-cold ethanol. After centrifugation, the pellets were air-dried for 1 h and resuspended with nuclease-free water (Invitrogen). RNA library was prepared using NEBNext Ultra II Directional RNA Libaray Prep Kit for Illumina by following the manufacturer's protocol.