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WISH was performed using a probe for Twist, used as a marker for migrating neural crest.
This was also confirmed using a probe for Slug, another neural crest-specific marker (Supplementary Fig. 2f).
A parallel analysis was performed using a probe for AldoA as a loading control and yielded comparable numbers to those obtained by normalization with RPS29 (data not shown).
FISH experiments using a probe for the entire chromosome 11 were performed on the same 11q13 cell line, grown under the same conditions.
Northern blotting using a probe for the 3' flanking region of the GSA target showed no signal in either the 35S GS-IR or the CoYMV:GS-IR transgenic line (Figure S4), although both exhibited unequivocal PTGS (Figure S2a, S2b).
In order to develop assays to more fully explore the potential heterogeneity of gene expression in RPCs, two ISH methods were used to examine expression, with an initial test using a probe for Cyclin D1, the gene expressed in the highest percentage of RPCs profiled.
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The following probes were used: a probe for miR-23b (5′-GGTAATCCCTGGCAATGTGAT-3′) and a scrambled control probe (5′-GTGTAACACGTCTATACGCCCA-3′).
A two-colour probe set was used, a probe for PIK3CA labelled with Gold 5 6 -carboxyrhodamine dUTP and a chromosome 3 centromere-specific probe labelled with green-fluorescein dUTP.
Cell pellets were resuspended in 1mM lysis buffer on ice for 10 minutes and sonicated using a probe sonicator for 30 seconds (three times, 10 seconds each).
We analysed this parasite population by Southern blot, using a probe diagnostic for the specific integration event.
Cells were selected with G418, and 96 resistant clones were screened by southern blot using a probe specific for the Flag-SMAD1 sequence.
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