title: RNAseq of S. stercoralis PV001 strain developmental stages
design: RNAseq of S. stercoralis PV001 strain developmental stages
library_name: L3_plus_g3
library_construction_protocol: Free-living stages grown in charcoal coprocultures; parasitic stages grown in Mongolian gerbils (See doi/10.1895/wormbook.1.134.1 for complete details) In noted samples, worms are isolated by the Baermann funnel technique and migrated through low melting temperature agarose to clean off residual debris and bacterial contamination.  Briefly, worms in fecal charcoal cultures are placed in an inverted glass funnel in a mesh-bottomed dish and surrounded with slightly warmer water.  Worms migrate into the warmer liquid and settle to the bottom of the funnel to which a rubber hose with a clamp is attached.  Worms are then collected, liquid removed, and mixed with low melting temperature agarose.  The worm and agarose mixture is allowed to gel and worms migrated out into a surrounding liquid at a slightly elevated temperature. For both L3+ and parasitic females, no agarose migration was performed; instead, worms were washed several times in media as noted in the individual samples.  (See doi/10.1895/wormbook.1.134.1 for complete protocol) TRIzol extraction of total RNA from samples snap-frozen in liquid nitrogen.  This method involves purifying the poly-A containing mRNA molecules from the total RNA using poly-T oligo-attached magnetic beads.  Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature.  The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers.  This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H.  These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters.  The products are then purified and enriched with PCR to create the final cDNA library for sequencing on the Illumina HiSeq 2000.  (See www.illumina.com Catalog # RS-930-2001 for a complete protocol)
attributes.tag: Experimental Factor: DevelopmentalStage
attributes.value: Tissue migrating third stage larvae