Le dataset qui va être utilisé au cours de ce TP provient d’une étude publiée par le laboratoire du Prof. Hanikenne. Si vous désirez le consulter, je vous invite à télécharger le PDF disponible dans l’onglet “downloads”
Ce jeu de données provient d’un séquençage ARN réalisé sur des racines d’individus WT ainsi que des individus mutants pour le gène FRD3. Ces différents génotypes ont été cultivés en hydroponie, soit à des concentrations normales en Zn (1 µM), soit en présence d’un excès de Zn (20 µM). Les racines ont ensuite été récoltées et les ARNs extraits au moyen du même kit que celui que vous utiliserez durant la seconde après-midi de TP.
Voici un extrait du Matériel et Méthodes de cet article décrivant le mode de culture ainsi que les paramètres de l’analyse de RNA-seq :
All experiments were conducted with Arabidopsis thaliana (Col-0) as the wild type and the Arabidopsis frd3-7 mutant (in the Col-0 background) (Roschzttardtz et al., 2011). Seeds were surface-sterilized and germinated on half-strength MS medium (Duchefa Biochimie, https://www.duchefa-biochemie.com) supplemented with sucrose (1% w/v; Duchefa Biochimie) and agar (0.8% w/v Select Agar; Sigma-Aldrich, https://www.sigmaaldrich.com). For hydroponics experiments, the seedlings were transferred 2 weeks after germination in hydroponic trays (Araponics, Belgiumhttp://www.araponics.com), grown for 3 weeks in control Hoagland medium, then submitted to experimental conditions for an additional 3 weeks. The nutrient solution was exchanged with fresh medium once a week and for the last 3 days before harvest. For plate experiments, seedlings were transferred 5 days after germination on solid Hoagland (0.8% w/v Select Agar in square plastic Petri plates; Greiner Bio-One, https://www.gbo.comGermany). The control Hoagland medium was modified as described by Talke et al. (2006) and Hanikenne et al. (2008), and included 1 µm Zn (ZnSO4.H2O), 5 µm Mn (MnSO4.H2O) and 10 µm FeIII-HBED [N,N-di-(2-hydroxybenzyl) ethylenediamine N,N-diacetic acid monohydrochloride]. Zn, Mn and/or Fe were omitted from the medium for deficiency experiments and were added as indicated for excess experiments. All experiments were realized under 8 h light (100 µm photon m–2 s–1, 22°C)/16 h dark (20°C) in a climate-controlled growth chamber. Unless otherwise stated, all experiments were conducted three times independently.
Upon harvesting, plant root tissues were blotted dry, immediately frozen in liquid nitrogen and stored at −80°C. Total RNAs were prepared using 100 mg of homogenized tissues and RNeasy Plant Mini kit with on-column DNase treatment (Qiagen, https://www.qiagen.com). Libraries for RNA-Seq were prepared from 1 µg of total RNAs with the TruSeq Stranded mRNA Library Prep Kit (Illumina, https://www.illumina.com), multiplexed and sequenced in two runs with an Illumina NextSeq500 device (high-throughput mode, 75 base single-end reads) at the GIGA-R Sequencing platform (University of Liège), yielding on average approximately 22 million reads per sample. Read quality was assessed using fastqc 0.10.1 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Quality trimming and the removal of adapters were conducted using trimmomatic 0.32 (Bolger et al., 2014), with the following parameters: trim bases with quality scores lower than Q26 in 5 and 3 of reads; remove any reads with Q < 26 in any sliding window of 10 bases; crop one base in 3 of all reads; and discard reads shorter than 70 bases. Overall, the quality filtering discarded between 7 and 9% of the raw reads. The Arabidopsis reference genome sequence (TAIR10) and annotation (201606 version) files were downloaded from Araport on 16 September 2016https://www.araport.org. Read mapping on the genome was achieved using tophat 2.1.1, with the following parameters: –read-mismatches 2; –min-intron-length 40; –max-intron-length 2000; 2 –report-secondary-alignments; –no-novel-juncs and providing an indexed genome annotation file. Raw read counts were obtained using htseq-count 0.6.1p1, and differentially expressed genes were identified by pairwise comparisons with deseq2 1.12.3 (Love et al., 2014). Genes were retained as differentially expressed when the log2 fold-change (FC) was >1 or <−1, with a Benjamini–Hochberg FDR adjusted P-value of <0.05. PCA plots were created with the PlotPCA function from r using rlog transformed data (Beginner’s guide, deseq2 package, 13 May 2014, http://www.bioconductor.org/packages/2.14/bioc/vignettes/DESeq2/inst/doc/beginner.pdf). GO enrichment analyses were conducted using the Thalemine tool on Araport (https://www.araport.org). The heat maps were constructed using the heatmap.2 function of the gplots r package.