title: GSM2445333: Adult Male Total RNA; Brugia malayi; RNA-Seq
library_construction_protocol: RNA was harvested using Trizol reagent. [Vaccinia Capping protocol] Total RNA from B. malayi microfilariae (25 μg) or adult males (1.5 µg) was incubated with 0.5 mM 3´ desthiobiotin-GTP, 0.1 mM S-adenosylmethionine (SAM) and 70 units of the Vaccinia Capping Enzyme in 1X Capping Buffer for 30 min at 37°C. RNA was then purified using the MEGAClear™ Transcription Cleanup Kit and eluted in 50 μL H2O. [Capture of 3' desthiobiotin capped RNA protocol] Capped RNA was mixed with 100 μL of hydrophilic streptavidin magnetic beads. The RNA-bead mixture was incubated for 20 min at room temperature, washed and RNA eluted with the addition of 1 mM biotin. The RNA, containing the 3´ desthiobiotin capped transcripts was collected and cleaned up using the RNA Clean and Concentrator Kit and eluted in 10 μL H2O and stored at −80°C until further use (‘Capped RNA’ sample). RNA libraries were prepared for sequencing using NEBNext RNA Ultra Prep Kit for Illumina