Muscle larvae (ML) of T. spiralis (strain ISS534) were obtained from rats 35 days post infection by digestion of minced skeletal muscle in 1% pepsin, 1% HCl for 3 h at 37°C with agitation as previously described [PMID:840494]. To purify adult worms and newborn larvae, Wistar rats at 6 weeks of age were inoculated with T. spiralis (strain ISS534) orally with a dose of 8000 larva per rat. At 30 h or 6 days post infection, all rats were executed and the entire intestines were removed, opened longitudinally and cut into pieces (about 0.5-1 cm). The fragmented intestine was put on a layer of gauze which was immersed into 37 °C 0.9% sodium chloride solution and incubated for 3 h. Adult T. spiralis worms would migrate into the liquid phase, which were harvested by centrifugation. To obtain new-born larvae, adult worms collected at 6 days post infection were incubated in IMDM in 75-cm2 cell culture plate at 37 °C, the newborn larvae were harvested every 12 h.
Extracted molecule
total RNA
Extraction protocol
Total RNA of T. spiralis (Ad, NBL and ML) was purified using Trizol reagent. 6 μg of total RNA from each preparation was treated with Oligo-(dT) conjugated magnetic beads to purify mRNA. Double-strand cDNA was synthesized guided by the Oligo-(dT) as a primer and then digested with the endonuclease NlaⅢ that recognizes the CATG sites. The Illumina adaptor 1, containing a MmeI restriction site, was added to the cDNAs attached to the magnetic beads, which was further digested with MmeI. Following MmeI digestion and dephosphorylation, cDNA fragments were purified and the Illumina adaptor 2 was ligated to the 3’ends of the tags to create tag library with different adapters at both ends. After15 cycles of linear PCR amplification, 95 bp fragments were purified from 6% TBE PAGE gels and attached to the Illumina sequencing chip for sequencing.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
cDNA
Instrument model
Illumina HiSeq 2000
Data processing
Basecalls performed using phred/phrap software. Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to Trichinella spiralis whole genome with SOAP2, allowing for no more than one nucleotide mismatch. Genome_build: Trichinella spiralis genome 3.7.1 Supplementary_files_format_and_content: tab-delimited text files include count for each sequence.
