Computational protocol: Systems-wide analysis of manganese deficiency-induced changes in gene activity of Arabidopsis roots

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Protocol publication

[…] Three independently grown batches of plants grown under Mn-sufficient and Mn-deficient conditions were used for analysis. For each sample, roots from 10 Mn-sufficient and 20 Mn-deficient plants were pooled and total RNA was extracted from roots or leaves using the RNeasy Plant Mini Kit (Qiagen), following manufacturer instructions. Nucleic acid quantity was analyzed with a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, USA). For RNA-seq, equal amounts of total RNA were collected and cDNA libraries for sequencing were prepared from total RNA following the manufacturer’s protocol (Illumina). The cDNA libraries were subsequently enriched by PCR amplification. The resulting cDNA libraries were subjected to sequencing on a single lane of an Illumina HiSeq 2000 machine. RNA-seq and data collection was done following the protocol of Mortazavi et al.. The length of the cDNA library was maintained from 250 to 300 bp with a 5′-adapter of 20 bp and a 3′-adapter of 33 bp at both ends. Eventually, the fragment length range of the cDNA was 200 to 250 bp. To quantify gene expression levels, 75-mers sequences were aligned to the genomic sequence annotated in TAIR10 using the BLAT program and RPKM (Reads Per Kbp per Million reads) values were computed using the RACKJ (Read Analysis & Comparison Kit in Java, software. […]

Pipeline specifications

Software tools BLAT, RACKJ
Databases TAIR
Application RNA-seq analysis
Organisms Arabidopsis thaliana
Diseases Radiculopathy, Manganese Poisoning
Chemicals Glucosinolates, Manganese