GSM4904571: Pseudomonas, 1 hr, rep A, batch 3; Lucilia sericata; RNA-Seq
RNA was extracted from 5 larvae from each sample. First, whole RNA was extracted using TriReagent, according to manufacturer's protocols. Each sample was macerated in 1 mL of cold TriReagent. Following this, 50 mL of ice-cold BAN reagent was added and the solution was vigorously mixed. Next, the tubes were centrifuged at 14,000 G at 4°C for 15 min to isolate the RNA from the DNA and proteins. Approximately 500 μL of the top clear layer was carefully removed with a pipet and added to 500 μL of ice-cold 100% isopropanol. The tubes were mixed via inversion three times and allowed to rest on ice for 10 min to precipitate the RNA. The precipitate was then centrifuged at 14,000 G at 4°C for 15 min. Next, the supernatant was completely removed, 1 mL of ice-cold 70% ethanol was used to wash the RNA pellet, and the pellet was centrifuged at 4°C for 5 min at 14,000 G. The ethanol was eluted, and any remaining ethanol was allowed to completely evaporate. The RNA was then dissolved in a 10μL mixture of 99 μL of DNase/RNase/Nucleotide-free water and 1 μL of SUPERase•In™ (Invitrogen). The extracted RNA was further purified using a Qiagen RNeasy Micro Kit and on-column DNase treatment following manufacturer protocols. RNA was eluted again into a fresh 1:100μL mixture of SUPERase•In and DNase/RNase/Nucleotide-free water and stored at -80°C until sequencing. TruSeq stranded mRNA-seq