TITLE: Mammalian Calling Cards Quick Start Guide
AUTHORS: Arnav Moudgil, Michael N. Wilkinson, Xuhua Chen, Robi D. Mitra

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Transposon calling cards can identify transcription factor (TF) binding sites. This involves fusing your favorite TF (YFTF) to the hyperactive </span><span style = "font-style:italic;">piggyBac</span><span> transposase (HyPBase). This is delivered to cells in conjunction with a </span><span style = "font-style:italic;">piggyBac</span><span> transposon. The TF will visit sites in the genome and YFTF-HyPBase will deposit transposons near binding sites. We then generate sequencing libraries to map the genome-wide localization of transposons. Finally, we identify significant clusters of insertions to identify TF binding sites.</span></div><style>
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[STEPS]
?. [Cloning and Sequence Validation of YFTF-HyPBase Fusions]
Make C- and N- terminal fusions of your favorite transcription factor (YFTF) with HyPBase. It is important to include a linker sequence between these genes. We strongly recommend the following amino acid linker sequence: KLGGGAPAVGGGPKAADK. We have tested many and have found this sequence works best. It is often convenient to make these fusion constructs by using In-Fusion (Clontech/Takara) or Gibson (NEB) cloning to drop YFTF into pRM1258/pENTR_myc-hypPBase. We do not have good antibodies to the piggyBac transposase, so we recommend designing your construct so that the chimeric protein is tagged with myc.
?. [Cloning and Sequence Validation of YFTF-HyPBase Fusions]
Validate the constructs. Perform restriction digest analysis on the plasmid with at least 3 restriction enzymes to make sure there were no gross rearrangements. Next, Sanger sequence the full chimeric gene, or alternatively perform Illumina sequencing on the whole plasmid. It is important to do the restriction digest and EITHER of the sequencing stratgies.
?. [Cloning and Sequence Validation of YFTF-HyPBase Fusions]
(Only required for Gateway strategy). Move the chimeric gene from the pENTR vector to an expression vector.
?. [Functional Validation of YFTF-HyPBase Fusions]
After creating the YFTF-HyPBase and HyPBase-YFTF fusions, the next steps are to validate them. First we will assess whether the fusions retain piggyBac transposase activity. We recommend transforming HCT-116 cells with the YFTF fusions and the BrokenHeart transposon along with appropriate controls. We recommend using Lipofectamine 3000 (following manufacturer's instructions) to deliver 1 µg total DNA to approximately 200,000 cells in each well of a 6-well plate. The following table summarizes each condition and the expected results.  ABCDEFGH1ConditionEmpty plasmidpRM1294 BrokenHeartpRM1114 CMV_HyPBaseYFTF-HyPBaseHyPBase-YFTFRed cells?Notes2Negative control0.5 µg0.5 µgNANANANone3Positive controlNA0.5 µg0.5 µgNANAMany4YFTF-HyPBaseNA0.5 µgNA0.5 µgNASomePerform in  duplicate5HyPBase-YFTFNA0.5 µgNANA0.5 µgSomePerform in duplicate6Optional control #1 -- a "lipofection only" negative control. This is not a bad idea, particularly if you are new to lipofections or are testing a new cell line and are concerned about toxicity. These cells should show high viability and no fluorescence signal. If these cells are viable but cells transfected with DNA are not, it may indicate issues with plasmid isolation (e.g. endotoxin contamination).Optional control #2 -- a GFP expression plasmid could be transfected in parallel to estimate overall transfection efficiencies.
ABCDEFGH1ConditionEmpty plasmidpRM1294 BrokenHeartpRM1114 CMV_HyPBaseYFTF-HyPBaseHyPBase-YFTFRed cells?Notes2Negative control0.5 µg0.5 µgNANANANone3Positive controlNA0.5 µg0.5 µgNANAMany4YFTF-HyPBaseNA0.5 µgNA0.5 µgNASomePerform in  duplicate5HyPBase-YFTFNA0.5 µgNANA0.5 µgSomePerform in duplicate6
?. [Functional Validation of YFTF-HyPBase Fusions]
The second validation will test whether the YFTF-fusions successfully redirect piggyBac insertions near YFTF binding sites. Since piggyBac inserts into TTAAs, we would like to be able to distinguish unique insertions into the same TTAA.  For this reason, we recommend 6 replicates per condition, at least for your two “test” samples, and the unfused piggyBac (1 6-well plate each, 3 total). In addition, we recommend running one well as a transposon-only negative control and one well as a mock lipofection negative control. Here we will use puromycin selection to obtain cells with transpositions. Once again, we will work with HCT-116 cells.  ABCDEFGH12ConditionEmpty plasmidpRM1304 PB_SRT_Rz_PuropRM1114 CMV_HyPBaseYFTF-HyPBaseHyPBase-YFTFAlive cells?Notes3No transfection control1 µgNANANANANoneNo colonies after selection4SRT only control0.5 µg0.5 µgNANANANoneNo colonies after a few days5Positive controlNA0.5 µg0.5 µgNANAManyPerform 6 replicates6YFTF-HyPBaseNA0.5 µgNA0.5 µgNASomePerform 6  replicates7HyPBase-YFTFNA0.5 µgNANA0.5 µgSomePerform 6 replicatesWe typically split each well 1:1 and transfer to a 10 cm dish after 24 hours. We add puromycin to a final concentration of 2 µg/ml after cells have seeded, typically 6-8 hours after transferring. Media is replenished every two days. All replicates are cultured separately. Cells are harvested after the SRT-only control transfectants are dead.
ABCDEFGH12ConditionEmpty plasmidpRM1304 PB_SRT_Rz_PuropRM1114 CMV_HyPBaseYFTF-HyPBaseHyPBase-YFTFAlive cells?Notes3No transfection control1 µgNANANANANoneNo colonies after selection4SRT only control0.5 µg0.5 µgNANANANoneNo colonies after a few days5Positive controlNA0.5 µg0.5 µgNANAManyPerform 6 replicates6YFTF-HyPBaseNA0.5 µgNA0.5 µgNASomePerform 6  replicates7HyPBase-YFTFNA0.5 µgNANA0.5 µgSomePerform 6 replicates
?. [Calling Card Library Preparation]
Calling card libraries can now be made from successfully selected cells. For first-time users, we recommend following the bulk calling card protocol for making libraries.
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?. [Sequencing, Analyzing, and Visualizing Calling Card Data]
If your library preparation has been successful, you are ready to sequence your calling card libraries. We have successfully sequenced libraries on the Illumina MiSeq, MiniSeq, HiSeq, and NextSeq platforms. Due to relatively low sequence complexity, we typically run our libraries with 50% PhiX genome spiked in.
?. [Next Steps]
If you've made it this far and your data look great, congratulations! One or both of the YFTF fusions have worked and successfully enriched for insertions near YFTF binding sites. You may now wish to repeat the above experimental workflow with your model systems, or try different transgenesis techniques (e.g. electroporation, nucleofection, viral transduction). Otherwise, you are now ready to move into your model system and study YFTF binding. Please let us know if you have any difficulties and we will do our best to provide assistance.
?. [Calling Card Library Preparation]
(Advanced) Depending on your application, you may also be interested in making single cell calling card libraries.
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