Computational protocol: Chromatin remodelling and antisense-mediated up-regulation of the developmental switch gene eud-1 control predatory feeding plasticity

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Protocol publication

[…] The presence and levels of gene expression were measured by whole-transcriptome sequencing (RNA-Seq) of Ppa-lsy-12(tu319) mutants and P. pacificus wild type. Culture populations were allowed to grow until their food was exhausted, immediately after which the cultures were processed for sequencing. Five mixed-stage plates were washed with 40 ml M9, centrifuged immediately at 1300 g for 4 min, rinsed with 40 ml 0.9% NaCl treated with 40 μl ampicillin and 40 μl chloramphenicol and shaken gently for 2 h, and finally concentrated into a pellet by centrifugation and immediately frozen in liquid nitrogen. NEBNext Ultradirectional RNA Library Kit (Cat # E7420L) was used to prepare libraries. RNA-Seq libraries were sequenced as 2 × 100-bp paired-end reads on an Illumina HiSeq 2000, yielding 30.6 million reads for the wild type and 31.6 million reads for the lsy-12 sample. Raw reads were aligned to the reference genomes of P. pacificus (Hybrid1) (www.pristionchus.org), using the software Tophat v.2.0.3 (ref. ). Expression levels were estimated and tested for differential expression using the programs Cufflinks and Cuffdiff v.2.0.1 (ref. ) resulting in 95 significantly differentially expressed genes including eud-1 (FDR corrected P value<0.01). The equivalent test for down-regulation of as-eud-1 was not significant despite no evidence of as-eud-1 expression in Ppa-lsy-12 animals, which is probably due to the reduced statistical power for differential expression detection resulting from the low expression of as-eud-1 even in wild-type animals. [...] All phenotypic data show Eu frequency calculated from total individuals screened. Total sample size is illustrated on graphs. Significant differences were tested by Fisher's exact test. For the expression data we performed Kruskal–Wallis test. All statistical analyses were implemented in the program Statistica v. 9 (Statsoft). […]

Pipeline specifications

Software tools TopHat, Cufflinks, Statistica
Databases Pristionchus.org
Applications Miscellaneous, RNA-seq analysis
Organisms Caenorhabditis elegans, Pristionchus pacificus
Diseases Xerostomia