all miRNA stem-loop RT primers (Table S6), and the second had 18s ribosome RNA primer (Table S7) for internal control [116]. The reverse transcription product was amplified using a miRNA- specific forward primer and a universal reverse primer on a Rotor- Gene 6000 (QIAGEN, Valencia, CA). Reactions were performed on three independent biological replicates. Quantitative Reverse Transcriptase PCR (qRT-PCR) of miRNA Target Gene Expression The expression of experimentally determined and computa- tional predicted target genes were assayed by qRT-PCR [11]. The reverse transcription product was amplified using gene-specific primers that generated amplicons that overlapped the known or predicted cleavage site (Table S8). Reactions were performed in triplicate on a Rotor-Gene 6000 (QIAGEN, Valencia, CA). Data was normalized to 18S RNA (Table S7) and analyzed using a comparative quantification procedure [115]. Only amplicons confirmed to be a single product by the melt curve from qRT- PCR were used for analysis. 59-RLM-RACE Validation of miRNA Targets Poly (A) mRNAs from the total RNA of maize roots were purified using mRNA purification kit (TaKaRa, Dalian, China). An RNA adapter was directly ligated to 100 ng of mRNA using a T4 RNA ligase (Invitrogen, Carlsbad, CA). Ligated mRNAs were reverse transcribed using Superscript III reverse transcription kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. 5’-RLM-RACE was performed on the selected maize targets [117]. Initial PCR reactions were done with an adapter primer and complementary gene specific primers. The nested PCR was performed with the nest adapter and the nest gene specific primers. The resulting PCR products were gel purified, cloned by TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced. Cluster Analysis of the Gene Expression A custom R clustering program was developed for cluster analysis. Two R packages ‘‘Cluster’’ and ‘‘mclust’’ were used for hierarchical cluster. ‘‘heatmap.20 from the R package ‘‘gplots’’ was used for drawing the heat map and the gene tree (Figure 5, 6, S2, S3, S4 and S5). Cis-regulatory Element Analysis of miRNAs Promoter Most miRNA genes, like protein-coding genes, are transcribed by RNA polymerase II (Pol II) that results in 59 capped and poly (A)-tailed transcripts called primary miRNAs (pri-miRNAs) [118,119,120]. Therefore, it is reasonable to assume that the promoters of miRNA genes might be located within the upstream region of pri-miRNAs. However, only the pre-miRNA sequence information is available in the public database such as miRBase [32]. To get TSS, the pre-miRNA coordinates of differentially expressed miRNAs were mapped to their B73 RefGen v1 chromosomal locations [27]. Only differentially expressed miR- NAs with known TSS were used for promoter analysis. Putative cis-regulatory elements were predicted only for the B73 inbred line due to availability of its reference genome. For the other two inbred lines Hz32 and Mo17, promoter sequences were not available due to lack of their respective genome assemblies. Cis- regulatory motifs were computationally predicted in the promoter region of 1000 bp upstream and 500 bp downstream with respect to TSS of the pri-miRNA genes. Search Tool for Occurrences of Regulatory Motifs (STORM) in a Comprehensive Regulatory Element Analysis and Detection (CREAD) suite of tools [121] was Characterization of Hypoxia Induced Maize miRNAs used to identify putative transcription factor binding sites using known experimentally derived position weight matrices from JASPAR core Plantae (http://jasper.genereg.net/) [63]. Custom scripts were developed to reduce false positive signal detections. The putative cis-regulatory elements those were over-represented (p-value ,0.001) in the promoter sequences of miRNAs as compared to random genomic sequences; and had a frequency of three or more occurrences per miRNA promoter sequences were considered. The p-value was obtained after adjusting for multiple testing by applying Bonferroni correction [122]. Cytoscape [80] was used for constructing and analyzing regulatory network. Supporting Information Figure S1 Size distribution of sequenced reads of control and 4 h waterlogging treated roots in Hz32. X- axis shows the size of the reads of the sequencing fragment. Y-axis shows the raw reads of each fragment. Red bar shows the results of control sample. Green bar shows the results of 4 h treatment sample. (TIF) Figure S2 Cluster 1 miRNA targets expression profile. The cluster was done on the basis of log2 (expression level in treatment/expression level in control). Yellow color shows down- regulation. Blue color shows up-regulation. (TIF) Figure S3 Cluster 2 miRNA targets expression profile. The cluster was done on the basis of log2 (expression level in treatment/expression level in control). Yellow color shows down- regulation. Blue color shows up-regulation. (TIF) Figure S4 Cluster 3 miRNA targets expression profile. The cluster was done on the basis of log2 (expression level in treatment/expression level in control). Yellow color shows down- regulation. Blue color shows up-regulation. (TIF) Figure S5 Cluster of novel miRNA targets expression profile. The cluster was done on the basis of log2 (expression level in treatment/expression level in control). Yellow color shows down-regulation. Blue color shows up-regulation. (TIF) Table S1 Grading of maize seedling of 3 maize inbred lines by phenotypic chlorosis under waterlogging stress. The grades from 1–5 show the tolerance of the maize inbred lines. Each grade has its own description. (XLSX) Table S2 Summary of small RNA sequencing data. Total reads are the raw reads from the sequencing library. Reads containing N are the reads containing uncertain nucleotide (N = A/T/C/G). Reads without N are the reads without uncertain nucleotide. Adaptor only reads are the reads that only have adaptor sequence. Reads with small insert are the reads length below 18 nt after removing the adapter sequence. Reads with large insert are the reads length over 26 nt after removing the adapter sequence. Filtered reads are the reads that passed all the filters described above. Unique sequences are the unrepeated sequence of the filtered reads. (XLSX) Table S3 Known miRNAs present at 4 h waterlogging and control treatment in Hz32 roots. First column is the miRNA id. The second column is the read per million (RPM) of PLoS ONE | www.plosone.org 13 June 2012 | Volume 7 | Issue 6 | e39786