Schematic diagram of RescueMu grid tagging and sequencing (RescueMu not to scale). Step 1: RescueMu is introduced into embryogenic callus followed by crossing of regenerated plants to active Mutator lines. Lines are screened for transposed RescueMu elements in plants lacking the original transgene array. Pollen from one RescueMu donor plant is crossed to multiple ears of a non-RescueMu line to generate tagging grids of up to 48 rows × 48 columns of trRescueMu plants in the field. Step 2: plant DNA prepared from pools of row or column leaves is used to generates transformed bacterial libraries of RescueMu plasmids. These are used as sequencing templates and for construction of a library plate representing the diverse insertion sites in grid plants. Step 3: genomic DNA is digested using two restriction enzymes (BamHI, BglII), religated into plasmids and transformed into E. coli. Step 4: after transformation, RescueMu plasmid-containing E. coli colonies are selected by plating onto carbenicillin agar plates and picked into 384-well plates with growth/freezing media. Overnight incubation is followed by a PCR reaction designed to amplify longer inserts with lengths up to 16 kb. Using the PCR product, eight 96-well sequencing plates (four for sequence from the left TSD and four from the right TSD) are created. Step 5: priming strategy and relative locations of PCR and sequencing primers within the RescueMu element. The sequencing reactions are read out from the TSDs to recover the germinal insert sequence. Although a BamHI and BglII double-restriction digest produces a shorter, easier-to-sequence insert length, it also increases the ambiguity in interpreting the sequence during analysis. Given successful sequencing in both directions, two GSS sequences may be submitted for every plasmid (sequence flanking the left and right TIRs). Two additional GSS sequences may be submitted for a plasmid when a BamHI, BglII or BamHI-BglII ligation site is encountered. Each of these occurrences yields sequence that was not necessarily contiguous in vivo. Dubious GSS sequences are designated with the suffix .1EL (re-created enzyme ligation site) or .2EL (re-created enzyme ligation of two restriction sites not encountered in vivo). Sequence flanking TIRs in vivo is submitted as GSS sequences with no suffix except the .x or .y (right or left) direction designation.