Simultaneous measurement of biochemical phenotypes and gene expression in single cells

Richer, Amanda L.; Riemondy, Kent; Hardie, Lakotah; Hesselberth, Jay R.

doi:10.1093/nar/gkaa240

Cited by 5 publications

(6 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Repair enzymes create nicks in the substrates, and sequencing quantifies repair activities by comparing modified and unmodified oligo substrates. Haircut profiles PARP-related single-strand repair, quantifying the activity of base excision repair and nucleotide excision/incision repair enzymes in a single reaction (See Figures 1-2 and Supplemental Figure 1 in (29), also (30)). We coupled Haircut with targeted transcriptome analysis (10X Genomics Pan-cancer capture panel, n=1,253 genes), allowing us to profile DNA repair activity and gene expression in individual cells.…”

Section: Resultsmentioning

confidence: 99%

“…MM134 cells were hormone-deprived in medium supplemented with charcoal-stripped FBS as previously described (59), then treated with vehicle (0.01% ethanol) or 100pM estradiol for 24hrs. After treatment, single cell suspensions were prepared according to 10X Genomics recommendations, and as described (29,30). Whole transcriptome and Haircut libraries were prepared as described (29,30), and separate targeted capture libraries were prepared according to the manufacturer’s instructions (https://www.10xgenomics.com/products/targeted-gene-expression).…”

Section: Methodsmentioning

confidence: 99%

“…After treatment, single cell suspensions were prepared according to 10X Genomics recommendations, and as described (29,30). Whole transcriptome and Haircut libraries were prepared as described (29,30), and separate targeted capture libraries were prepared according to the manufacturer’s instructions (https://www.10xgenomics.com/products/targeted-gene-expression). Supplemental File 2 includes workflow information and normalized gene expression data for the targeted capture panel and Haircut probes, with single-cell annotation for clustering, cell cycle prediction, and ER target genes scores, and is available at: https://osf.io/rw2n6.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Co-regulator activity of Mediator of DNA Damage Checkpoint 1 (MDC1) is associated with DNA repair dysfunction and PARP inhibitor sensitivity in lobular carcinoma of the breast

Sottnik,

Shackleford,

Richer

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Invasive lobular carcinoma of the breast (ILC) are typically estrogen receptor α (ER)-positive and present with biomarkers of anti-estrogen sensitive disease, but growing laboratory and clinical data, including poor long-term outcomes faced by patients with ILC, suggest endocrine response and ER function are unique in ILC. We previously identified the DNA repair protein Mediator of DNA Damage Checkpoint 1 (MDC1) as an ILC-specific ER co-regulator necessary for ER genomic activity, and that MDC1 co-regulator activity was associated with dysfunctional canonical DNA repair roles of MDC1. To understand these potentially reciprocal activities of MDC1 in ILC, we profiled the MDC1 interactome and found that MDC1 associated proteins in ILC cells mirror a “BRCA-mutant” state lacking MDC1 interaction with key homologous recombination (HR) proteins. Single-cell gene expression and DNA repair activity showed that specific activation of ER:MDC1 target genes was associated with increased PARP-associated DNA repair and decreased HR gene expression. These data suggest that HR is dysfunctional in ILC, which was supported by a lack of DNA damage-induced RAD51 turnover in ILC cells, and an elevated DNA damage response protein signature in a subset of ILC tumors. We tested whether this HR dysfunction could be exploited using PARP inhibition, and found that talazoparib treatment produced a durable growth suppression bothin vitroand in ILC cell line xenograftsin vivo. The ILC-specific ER:MDC1 association creates a new context for ER and MDC1 function in ILC, at the cost of a DNA repair dysfunction that may be therapeutically targetable.SignificanceILC are rarely associated with biomarkers of overt HR deficiency, as such patients are rarely eligible for treatment with PARP inhibitors. Our work suggests ILC present with a previously unappreciated form of HR dysfunction, linked to ILC-specific genomic activity of ER, that imparts sensitivity to PARP inhibition.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Co-regulator activity of Mediator of DNA Damage Checkpoint 1 (MDC1) is associated with DNA repair dysfunction and PARP inhibitor sensitivity in lobular carcinoma of the breast

Sottnik,

Shackleford,

Richer

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Available in flexible wrapper functions, both reference building and new classification can be directly achieved through scRNA-seq objects at hand, without going through format conversions or manual extraction. The wrappers can also be expanded to other single cell RNA-seq object types, including the HDF5-backed loom objects, as well as other data types generated by CITE-seq and similar experiments 31 . Tutorials are documented online to help users integrate clustifyr into their workflows with these and other bioinformatics software.…”

Section: Discussionmentioning

confidence: 99%

clustifyr: an R package for automated single-cell RNA sequencing cluster classification

Gillen

Sheridan

et al. 2020

F1000Res

Self Cite

View full text Add to dashboard Cite

Assignment of cell types from single-cell RNA sequencing (scRNA-seq) data remains a time-consuming and error-prone process. Current packages for identity assignment use limited types of reference data and often have rigid data structure requirements. We developed the clustifyr R package to leverage several external data types, including gene expression profiles to assign likely cell types using data from scRNA-seq, bulk RNA-seq, microarray expression data, or signature gene lists. We benchmark various parameters of a correlation-based approach and implement gene list enrichment methods. clustifyr is a lightweight and effective cell-type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr

show abstract

“…To characterize the heterogeneity of DNA repair cellular activities, Richer et al have performed simultaneous measurement of mRNA transcripts and DNA repair mechanisms. 71 They have developed a technique called single-cell haircut (sc-haircut), to compartmentalize single cells with barcoded oligo-dTs and enzymatic substrates for DNA repair using droplet microfluidics (Fig. 2).…”

Section: Combining Transcriptomic and Genomic Analysismentioning

confidence: 99%

Frontiers in single cell analysis: multimodal technologies and their clinical perspectives

et al. 2022

View full text Add to dashboard Cite

show abstract

Simultaneous measurement of biochemical phenotypes and gene expression in single cells

Cited by 5 publications

References 24 publications

Co-regulator activity of Mediator of DNA Damage Checkpoint 1 (MDC1) is associated with DNA repair dysfunction and PARP inhibitor sensitivity in lobular carcinoma of the breast

Co-regulator activity of Mediator of DNA Damage Checkpoint 1 (MDC1) is associated with DNA repair dysfunction and PARP inhibitor sensitivity in lobular carcinoma of the breast

clustifyr: an R package for automated single-cell RNA sequencing cluster classification

Frontiers in single cell analysis: multimodal technologies and their clinical perspectives

Contact Info

Product

Resources

About