Similar protocols

Pipeline publication

[…] .0 Fluorometer (Life Technologies catQ32866). The cDNA was diluted to a final concentration suitable for monoclonal amplification by emulsion-PCR, while copies of template molecules were attached to beads. Beads were deposited onto slides upon which the sequencing reactions, as well as washing steps, were performed automatically in the 5500 SOLiD platform. The 5500 SOLiD sequencer was used to read short DNA sequences (50nt long), from the DNA attached to the beads with 50 rounds of oligonucleotide annealing and ligation (five different primers, 10 sequential ligations per primer)., Reads in color-space format were mapped against the fifth version of the S. mansoni reference genome [], using Bowtie aligner [] with adjusted parameters []. Reads that mapped to ribosomal and globulin sequences were excluded from further analysis; only uniquely mapped reads were considered and used for downstream analysis., Differential expression was assessed by using transcript abundance count tables as input to the DESeq2 package [] in the R environment []. Differentially expressed transcripts were analyzed among replicates of the libraries generated from the 3 hour cultures against those of the 12 hour cultures of the same serum (peripheral or portal). Transcripts with adjusted P values lower than 0.05 were considered differentially expressed. In the final list of differentially expressed transcripts, one was considered up-regulated only if they exhibited a Log2Change fold higher than 1, and down-regulated if Log2Change fold was lower than -1 []., GO enrichment analysis was performed with Blast2GO, software available at https://www.blast2go.com/blast2go-pro/download-b2g., The relative expression of a subset of the genes found deferentially expressed between SPO3H and SPO12H was determined with RT-qPCR. Primer 3 software [] was used to design the primers and probes for each gene. The oligos were acquired from Thermo Fisher Scientific (Custom TaqMan Gene Expression Assay), Brazil (sequences available in ). First strand cDNA was synthesized from 1μg of total RNA samples by using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Brazil—cat4368814) according to the manufacturers recommendations. The qPCR reactions were performed using a ViiA 7 Real-Time PCR System, with 384-well block equipment and TaqMan Univers […]

Pipeline specifications

Software tools Bowtie, DESeq2, Blast2GO
Organisms Schistosoma mansoni, Spirometra erinaceieuropaei, Mesocricetus auratus
Diseases Disease, Infection, Trematode Infections
Chemicals Nucleotides