Question for clonmapper protocol

[litingrubmgo]

Dear brocklab research group： You did an excellent job in the field of lineage-tracing. Our research group is using the technology you developed to construct the ClonMapper library.In order to guarantee the quality of barcodes, we hope to obtain high quality bar codes. 1.Would you mind telling us the company that you ordered the barcode-sgRNA? You can provide the company/product links if it is convenient.

2. I also want to know how to confirm the quality of the barcode-sgRNA（Maybe 20-bp random sequence includes the cleavage site/Maybe the size of the barcode is not stable）.Do I need to think about it? Or we just order it directly from the company. There is no need to think about it.
3. I'm also confused about this software： https://github.com/boutroslab/cld/
   Is it used to design“20-bp random sequence”? I am looking forward to your reply.Thank you so much！

[daylinmorgan]

Hope to address each of your questions below.

1. Barcode oligos for plasmid generation were ordered from IDT with the sequences found in Table 1 (see CROPseq-PrimeF-BgL-BsmBI and CROPseq-RevExt-BgL-BsmBI).
2. You can confirm the successful generation/diversity of you plasmid library with targeted sequencing as described in SgRNA Barcode Sampling. The size of the barcode is stable. There is a rare chance sgRNA-barcodes within the parental plasmid library are a base or two smaller or larger than 20 bp. However, they are not likely to change once stably integrated.
3. I don't know anything about this software as our barcodes are N(20) random sequences and not designed.

Are you trying to use the gRNA's to target specific sequences? If so, these would replace the N(20) sequence within CROPseq-PrimeF-BgL-BsmBI.

[litingrubmgo]

Thank you for your kindness in answering my questions. Your answer successfully solved my problems. Now，I still have two questions. 1.In “sgRNA Barcode Library Plasmid Pool Assembly” Part，why did you choose T4 ligase buffer（not T7 DNA Ligase Reaction Buffer）and T7 ligase to ligate gRNA barcode DNA into transfer vector？ 2.After we finish electroporation，we need to use “Recovery Media”. We do not know if the “Recovery Media”is “SOC Medium”？Would you mind providing product information ？[image: image.png]

Thank you for your prompt reply. Best regards Ting Li

On Mon, May 15, 2023 at 20:50 daylin @.***> wrote:

Hope to address each of your questions below.

1. Barcode oligos for plasmid generation were ordered from IDT https://www.idtdna.com/pages with the sequences found in Table 1 (see CROPseq-PrimeF-BgL-BsmBI and CROPseq-RevExt-BgL-BsmBI).
2. You can confirm the successful generation/diversity of you plasmid library with targeted sequencing as described in SgRNA Barcode Sampling https://docs.brocklab.com/clonmapper/single-page-protocol/#sgrna-barcode-sampling. The size of the barcode is stable. There is a rare chance sgRNA-barcodes within the parental plasmid library are a base or two smaller or larger than 20 bp. However, they are not likely to change once stably integrated.
3. I don't know anything about this software as our barcodes are N(20) random sequences and not designed.

Are you trying to use the gRNA's to target specific sequences? If so, these would replace the N(20) sequence within CROPseq-PrimeF-BgL-BsmBI.

— Reply to this email directly, view it on GitHub https://github.com/brocklab/clonmapper/issues/1#issuecomment-1548384101, or unsubscribe https://github.com/notifications/unsubscribe-auth/A7IXRMOXNEVJ22FWLFQAYHLXGJ3JDANCNFSM6AAAAAAXDFNUKM . You are receiving this because you authored the thread.Message ID: @.***>

[daylinmorgan]

In “sgRNA Barcode Library Plasmid Pool Assembly” Part，why did you choose T4 ligase buffer（not T7 DNA Ligase Reaction Buffer）and T7 ligase to ligate gRNA barcode DNA into transfer vector？

This is a historic protocol in the lab. We suspect that there would be a negligible difference between T7 ligase buffer and T4 ligase buffer. It may just be that this was the buffer we had on hand when originally generating the libraries.

2.After we finish electroporation，we need to use “Recovery Media”. We do not know if the “Recovery Media”is “SOC Medium”？Would you mind providing product information ？

Yes, SOC Medium, we typically use this one.

[litingrubmgo]

Thank you for your answer again. Your reply is like a light that illuminates my heart！now，I still have problems about this protocol.

1.In your paper“Multifunctional barcoding with ClonMapper enables high-resolution study of clonal dynamics during tumor evolution and treatment”. you use electrocompetent SURE 2 cells (Agilent,200152) as transformation material.But we check the product number. It uses for chemical transformation. Are you sure it can be used for Electroporation？or would you mind give us right product information.

2.In the “3.8 Recall and Isolation of Barcoded Lineages” part of protocol.It said“Set sort gate on GFP and BFP double positive gate indicative of a recalled cell”. We check the plasmid you use，Cropseq-BFP-WPRE-TS-hU6-BsmbI {Addgene # 137993 / Brock LabAA112} ，which includes “tag BFP” not “BFP”. Recall-miniCMV-sfGFP includes “sfGFP”. My quesiton ：The fluorescent proteins of sfGFP and GFP have different excitation and emission wavelengths，“tag BFP” and “BFP” also have similar problem.How can you sort the recallled cells. Is it OK for you to provide the detail of parameters you used for sorting？ Do you use BD FACSAria Fusion？

3.The GFP and BFP signal overlap , will it affect the isolation of Barcoded cells of interest?

I am looking forward to your reply!

Best regards Ting Li

[daylinmorgan]

1.In your paper“Multifunctional barcoding with ClonMapper enables high-resolution study of clonal dynamics...

You can use Endura Electrocompetent Cells.

2.In the “3.8 Recall and Isolation of Barcoded Lineages” part of protocol.It said“Set sort gate on GFP and BFP double positive gate indicative of a recalled ...

The plasmid information is correct the proteins are tagBFP and sfGFP. I don't have specific parameters to provide as we have used several different sorters. Perhaps you could speak with someone at your institution (or cytometry core) regarding sorting a double positive population.

3.The GFP and BFP signal overlap , will it affect the isolation of Barcoded cells of interest?

There should be minimal overlap between the signals. If you are concerned you can use compensation controls to correct for it.

[litingrubmgo]

Sorry to bother you again！ I want to know how many reads you suggest to be collected as a minimum to determine the complexity of the library（sgRNA Barcode library sequencing）. Thank you again for your help！

[daylinmorgan]

You can reference panels C and D in Extended Data Figure 1 here.

Typically we sequence at a depth exceeding 100 million reads to assess overall plasmid diversity. You can first perform a shallow sequencing if you prefer before sequencing more deeply to estimate what would be necessary to approach sequencing saturation.