Figure 1.
Reprogramming from leaf cells to chloronema apical cells.
(A) Upper parts of gametophores. (B–D, F–P) Dissected leaves after indicated time. (E) Intact leaf. Bars in A: 1 mm; B: 1 mm for B–D and I–L; E: 0.2 mm for E–H and M–P.
Figure 2.
Workflow for 5′-DGE library preparation.
(a) 1st strand cDNA is synthesized from mRNA. (b) At the 5′ end of the mRNA, cDNA synthesis continues onto the DNA/RNA chimeric oligonucleotide. (c) Three-cycle PCR is performed to produce the double-stranded cDNA. (d) the double-strand cDNA fragments are digested by EcoP15I. (e) Digested P2-attached 5′ tag fragments are captured by streptavidin-magnet beads and ligated with P1 adapter. (f) 5′-DGE library is amplified, and 97 bp fragments are purified after PAGE.
Figure 3.
Qualitative and quantitative nature of the 5'-DGE tags.
(A) Tags mapped to reverse and forward strands, representing forward- and reverse-strand transcripts, are shown in the upper panel and lower panel, respectively. Exons and introns of three gene models are shown by black boxes and horizontal lines. The values given on the left define the level of raw tag/normalized tag (genome average = 1). (B) Scatter plot of technical replicates (test1 and test2) in tags per million (TPM). (C) Scatter plot of biological replicates (rep1 and rep2). (D) Correlation between tag counts and the copy number per 0.1 ng polyA RNA measured by qRT-PCR (test1 sample). (E) Correlation of fold changes measured by SOLiD-DGE tags and by qRT-PCR. The ratios between 0 and 3 h samples were compared.
Figure 4.
SOM-based clustering analysis for time-course samples.
3,956 genes with significant expression changes were clustered into 3 by 3 groups with a SOM algorithm. Relative expression changes for each gene are shown in the corresponding cluster. Green lines in individual clusters indicate the average calculated from the relative expression levels of the genes. Line color corresponds to the relative expression level at 0 h (scale to the right).
Figure 5.
Expression patterns of transcription factors, epigenetic-related and reprogramming-related genes.
(A) Heat map representation of expression patterns during reprogramming for reprogramming-related genes. Numbers in parentheses indicate JGI protein identifiers. (B) Expression patterns of transcription factor genes sorted according to gene expression pattern tree. Four distinct clusters are identified to the left. (C) Expression patterns of epigenetic-related genes.