Computational protocol: Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo

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Protocol publication

[…] The raw data (fastq) were trimmed for Phred Quality Score > 20, read length > 30 bases, and the ribosome sequences were removed with tool sortMeRNA []. The mapper Bowtie version 2 [] was used to align reads against the A. thaliana transcriptome (with ‘local’ option and other default parameters). The 33602 annotated genes were extracted from TAIR (v10) database corresponding to the representative gene model (longest CDS) given by TAIR. The abundance of each gene was calculated by a home-made script which counts only paired-end reads for which both reads map unambiguously one gene, removing multi-hits.All the statistical analyses were done with the R software using also EdgeR package version 3.8.6 []. For the pilot experiment, to compare the gene expression, the raw counts were normalized to take the difference of the library sizes into account with TMM method and a sequencing date effect. It was done with a negative binomial generalized linear model with one factor (sequencing date). Normalized counts equal to the raw counts divided by the scaling factor minus a date sequencing effect. The second experiment concerned the tissue comparison between epidermis and mesophyll. First, genes which did not have at least 1 read after a count per million normalization in at least one half of the samples, were discarded. Then, raw counts were normalized using TMM method and count distribution was modelled with a negative binomial generalized linear model where the tissue type and the harvest date were taken into account and where the dispersion is estimated by the edgeR method. A likelihood ratio test was performed to evaluate a tissue effect. Raw p-values were adjusted with the Benjamini–Hochberg procedure to control the False Discovery Rate (FDR). A gene was declared differentially expressed if it’s adjusted p value ≤ 0.05. […]

Pipeline specifications

Software tools SortMeRNA, Bowtie, edgeR
Databases TAIR
Application Metatranscriptomic sequencing analysis