RNA-seq of Brugia malayi adult female body (worm 1)
Individual adult female B. malayi were washed 3x in nuclease free PBS then dissected using Eliminase-cleaned 0.15 mm minuten pin dissecting probes in PBS in a petri dish under a ZEISS Stemi 508 with Sony Exmor CMOS IMX178 camera. For head vs body RNA-seq, the head was severed by crossing two probes adjacent to the vulva. Individual tissues were transferred to 2 ml Safe-Lock tubes containing 300 l TRIzol LS and 100 l nuclease free water, vortexed, flash frozen in liquid nitrogen, and stored at -80C. For RNA extraction, samples were thawed and homogenized with a single 3 mm Eliminase washed stainless steel ball bearing in for 3 min at 30 hz (TissueLyser II, Qiagen) then purified by the Direct-zol RNA microprep kit (Zymo, #R2060).  The Clontech SMARTSeq v4 Ultra-Low Input RNA kit was used with 1.7 ng of input RNA from each sample, as determined by Agilent RNA 6000 Pico Kit on a 2100 Bioanalyzer. Full length cDNA was quantified by 2100 Bioanalyzer. 150 pg of amplified cDNA was tagmented and index-amplified using Nextera XT adapters. DNA quantity was assessed by Qubit (dsDNA HS Assay Kit) and quality by 2100 Bioanalyzer. Libraries were then balanced for a single lane of 1x100bp sequencing on Illumina HiSeq 2500.