RNA-seq of Brugia malayi adult female cryosection (worm 6)
Individual adult female B. malayi were picked into 0.005% methylene blue in RPMI 1640 with L-glutamine for 2 min then washed 3 x in RPMI 1640. Stained worms were picked using Eliminase cleaned 0.15 mm minuten pin dissecting probes directly into clear TissueTek O.C.T. in a Stainless Steel Base Mold. The mold was positioned immediately prior to staining on a bed of dry ice under a ZEISS Stemi 508 with Sony Exmor CMOS IMX178 camera. Once in O.C.T., the body was straightened so that the head region was positioned parallel to the long face of the mold using the probe. A ~0.5 l methylene blue (0.1% in water) dye dot was then placed roughly 1 mm above the anterior-most portion of the head to act as a location marker. During freezing the position of the worm was imaged in relation to the edge of the mold and the dye marker to calculate distance between the block edge and the dye dot as well as the dye dot to the sample. Frozen O.C.T. blocks were covered in parafilm, indexed, and stored at -80C until sectioning. Cryosections (48 x 20 m) along the anterior-posterior axis were taken on a Leica CM3050 S Research Cryostat by positioning O.C.T. blocks perpendicular to the cutting face. The number of sections required to pass through the dye dot was used to estimate the number of sections required to reach the worm head based on images taken during embedding. A 40x loupe was used to interrogate the O.C.T block for signs of the methylene blue stained sample. Edges of the O.C.T block were shaved to minimize O.C.T. contamination and sections were kept individually on dry ice in chilled 1.5 ml tubes until RNA extraction. RNA was extracted by thawing sections in 75 l nuclease free water for 30 s, followed by pipette mixing in 225 l TRIzol LS, then purified with the Direct-zol 96 kit (eluted in 20 l of nuclease free water).   The library was prepared using 8 l of RNA for steps 2.1 through to 2.11.11 of the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina using NEBNext Multiplex Oligos for Illumina. Libraries were sequenced on an Illumina Novaseq 6000 (2x150bp, S1 flow cell).