Quantitative Real-Time PCR (qRT-PCR) validation of ATH1 arrays A set of 23 DEGs from microarray analysis of wus mutants were selected for validation by qRT-PCR. Primers were designed using Quantprime (Arvidsson et al 2008) to amplify 175-250bp fragments spanning introns (Table S.I. 1 below). Total RNA was isolated using the RNA Easy kit (Qiagen) and treated with DNase I (MBI Fermentas). First strand synthesis was performed using First Strand cDNA synthesis kit (MBI Fermentas). The qRT-PCR was performed using CFX96 Real-Time PCR detection system (Bio-Rad Laboratories) with iQ SYBR Green supermix (Bio-Rad Laboratories) and analyzed with CFX Manager software (Bio-Rad Laboratories). Reactions were carried out in triplicate. Melt-curve analysis was performed to confirm specificity of the amplification reaction and normalization was performed with primers specific for eukaryotic initiation factor 1a (At5g60390) and actin-2 (At3g18780). The majority of DEGs showed similar differential expression in both microarray and qRT-PCR analysis (Figure S.I. 1 below). Gene Id AT1G15040 AT1G15040 AT5G19170 AT5G19170 AT2G20180 AT2G20180 AT1G11350 AT1G11350 AT5G39520 AT5G39520 AT1G62320 AT1G62320 AT4G21200 AT4G21200 AT5G55470 AT5G55470 AT5G04230 AT5G04230 AT2G05160 AT2G05160 EF1a EF1a ACTIN ACTIN At1g15040 At1g15040 At1g51960 At1g51960 At3g26900 At3g26980 At2g15790 At2g15790 At2g37860 At2g37860 At1g77740 At1g77740 At4g30950 At4g30950 At1g04230 At1g04230 At3g08010 AT3G08010 At5g52330 At5g52330 At5g53810 At5g53810 Primer Forward/Reverse (F/R) F R F R F R F R F R F R F R F R F R F R F R F R F R F R F R F R F R F R F R R F F R F R F R Sequence GGC TGC GCT TTA GTT TAT CAG GAG AGC CTC TAG AAA CTC AGC TTG GG TCG CTC AGA TTT GGC TGG CAT C CCA TCC GCC GTC CAA TAT GTA AAC ACT TAT ACC TCG CTG CAA CAA GTC GTT GCA TGC CCG GAT ACA TCA TTG CAA AGC TGC AAC ACC GGA ATC TC TCT TTG CTT CAC CGG GTC AAA TAG TGA CGA AAT GGG AGC TTC AAG GG TCT GGT ACA AGC GCG AGA ACT G AGC TAT GGA TCC GGG ACA GAT TG ACC CGC ACT CTC ATA CTT CTG ATG TGG TGA CTT ATT TCA GGC ATG GAG TAA CGG CGT CGT ATG ATG GAC AC ACG AGC AGC ATT TGT CTT TCC AC AAC ACC GGA GTA TGT GAA CTG C TTG ATG ACC CAA GCA GCC TCA C AAT TTC AGC TCG GCC TCG AAC G TTA CTC CTA CCT CGG CTT CTC C CAA ATG CGG AGC AAG AGG AAC A TGA GCA CGC TCT TCT TGC TTT CA GGT GGT GGC ATC CAT CTT GTT GGT AAC ATT GTG CTC AGT GGT GG GAC CTG CCT CAT CAT ACT CG GGA AGA CCA CCA CAC TCG TC GTG GAA GGA AAG CAT ATC AGA CAG C CAT CAC GAA GGA GGA GCG G GTT GTA CTC TTT GTT GAG GCT GCG TTC ATG ACT GTT GCA TCT CTC AAG G GCA GCA TAC ACA GTG ACT CTT CC CCC CAC CTT GTA TCA TAA ACC CC CTC TTT TGA ACT TCA GCA ACA GTG GC GAA GCT GCT AAG AAC TAT GGG ATC CG CTC TTT TGA ACT TCA GCA ACA GTG GC CTT GTG GAC GTC GCA GTG AGA G CTG TTG CAA CAA AGT GTC AAA ACT AG GCT TCT TCT GCT CGT GTT TCT CC TTC TGC ACT GTC AGC TGA AGG CGG AAA TGG ATA TCT TGC TGC TTC GGA TTG CGC GTG GAG AAA AAA CC CAT TGT TAC CAT CAC TCA AGA CTT GG CAC AGT GTC ATA ACG TTC TTG CAA CC GTGGACCGACCTTTCTTCCTTG CACCGAAGCTTGTCCATTTGGC GAGGACTTGGCAACACACTAGGTC CCACATGGTTCACACCGGGATAAG Table S.I. 1. Primers used for qRT-PCR validation of array data. Figure S.I.1. A set of 23 genes showing statistically significantly increased or decreased expression between wus GABI-KAT mutant (GABI_870H12) and wild type in the ATH1 arrays were selected for validation by qRT-PCR. The graph represents a scatter plot of Log 2 ratios of expression in the wus mutant and wild type after 30 hrs treatment with the cytokinin 2iP. qRT-PCR values (X axis) and ATH1 array values (Y-axis) are plotted against each other. Most genes show a strong correlation between the two data sets, either in up-regulation (upper right quadrant) or down-regulation (lower left quadrant). Characterizing wus mutants. The RNA product produced by the SAIL and GABI-KAT alleles of WUS was examined using semiquantitative RT-PCR. Primers 63F(CATCACGGTGTTCCCATGC) and 23R(GACCAAAC-AGAGGCTTTGCT) spanning the insertion site amplified a 310bp product from wild type CDNA after 30 hours of 2iP induction, when WUS transcriptional reporters are expressed within transdifferentiating LRP. Whereas no product was amplified from wild type samples at the 0 hour time point (when no WUS reporter expression is observed), or from homozygous mutants for either of the wus alleles at both time points. Conversely primers Wus 11F (CAAGCTCAGGTACTGAATGTGG) and Wus7R (CCATACTTCCAGATGGCAC), which were both located downstream of the insertion sites, amplified a 320bp product in both wus mutants after 0 and 30 hours 2iP. Whereas the same primer combination only yielded this product in the 30 hour 2iP sample of wild type and not the 0 hour sample. Primers for housekeeping genes UBC (F:TCAAATGGACCGCTCTTATC, R:CACAGACTGAAGCGTCCAAG) and Ran1(F:GTCATTGTTGGTGATGGAGGC, R: CATTCTTGTATGTGAGCCGTGC) were used as controls. Fully quantitative PCR showed a 50 fold higher amplification for the same primers in the mutants compared to wild type. These results suggest that there is increased expression of a truncated RNA product in the wus mutants even in the absence of a cytokinin treatment necessary for expression of WUS transcriptional markers in the root of wild type plants. Similar mis-expression of RNA products adjacent to insert sites, driven by TDNA promoter sequences, has been previously reported (Ulker et al. 2008). To understand the T-DNA insertion features of the GABI-KAT wus mutant (GABI_870H12) a left border (LB) primer (ATATTGACCATCATACTCATTGC) and GABI-KAT Right border (RB) primer (GTGGATTGATGTGATATCTCC,) were used to amplify flanking sequences from GABI-KAT mutant. The right and left primer (RP: TAGATGCAAATTTGGTTTGCC & LP: AAAACCTTCTTTTTGGGTCCC) flanking the TDNA insert as given by the GABI-KAT database was used. While LB and RP primer amplified a product of expected size, RB and LP gave none suggesting aberrant TDNA insertion. When the LB was used with LP an unexpected product of 750bp size was amplified in the GABI-KAT mutant and not in the controls. This suggests an inverted duplication at the insertion location, with cis-elements within the insert driving transcription of a truncated wus product. Ulker, B., Peiter, E., Dixon, D.P., Moffat, C., Capper, R., Bouche´, N., Edwards, R., Sanders, D., Knight, H. and Knight, M.R. (2008) Getting the most out of publicly available T-DNA insertion lines. Plant Journal, 56, 665-677.