Illumina HiSeq 2000 paired end sequencing; RNAseq of S. stercoralis PV001 strain L3i activation
RNAseq of S. stercoralis PV001 strain L3i activation
DA_20130516
S. stercoralis PV001 strain were isolated from seven-day-old charcoal coprocultures (incubated at 25 degrees C) by the Baermann technique at 27 degrees C.  L3i were subsequently washed twice in deionized water and incubated in M9 buffer supplemented with antibiotics for three hours at room temperature. (See doi: 10.1895/wormbook.1.134.1 for complete protocol) An aliquot of untreated L3i (no stimulation) were pelleted and frozen in 100 ul TRIzol reagent.  Subsequently, L3i were distributed amongst the following conditions, each supplemented with antibiotics: M9 buffer, DMEM (supplemented with L-glutamine, 4.5 g/L glucose, and sodium pyruvate), 200 uM 8-bromo-cyclic guanosine monophosphate (cGMP) in M9 buffer, and 400 nM Delta7-dafachronic acid (DA) in M9 buffer.  L3i were incubated at 37uC and 5% CO2 in air for a total of 24 hours using 500 L3i in 100 ul of liquid in each well of a 96-well round-bottom plate with 24 wells per condition.  L3i from 23 wells were pooled and the pellet frozen at -80 degrees C in 100 ul TRIzol reagent.  This protocol was repeated for a total of four biological replicates. Extraction in TRIzol Reagent per the manufacturer protocol. (See www.lifetechnologies.com) Libraries were constructed using the Illumina TruSeq RNA Sample Preparation Kit v2.  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 http://www.illumina.com/products/truseq_rna_sample_prep_kit_v2.ilmn)