Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry

Wear, Emily E.; Concia, Lorenzo; Brooks, Ashley; Markham, Emily A.; Lee, Tae-Jin; Allen, George C.; Thompson, William F.; Hanley‐Bowdoin, Linda

doi:10.1007/978-1-4939-3142-2_6

Cited by 14 publications

(32 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells from eight cultures were 660 combined for each of three biological replicates. 661 Nuclei were isolated as described previously (Lee et al, 2010;Wear et al, 2016) with the 662 addition of a Percoll gradient step. The frozen cell pellet was ground at 4°C in 40 mL of cell lysis 663 buffer (15 mM Tris-HCl pH 7.5, 2 mM EDTA, 80 mM KCl, 20 mM NaCl, 15 mM β-664 mercaptoethanol, and 0.1% Triton X-100) using a commercial blender.…”

Section: Arabidopsis Cell Culture and Nuclei Isolation 652mentioning

confidence: 99%

See 1 more Smart Citation

Genome-Wide Analysis of the Arabidopsis Replication Timing Program

et al. 2018

Self Cite

View full text Add to dashboard Cite

33Eukaryotes use a temporally regulated process, known as the replication timing program, to 34 ensure that their genomes are fully and accurately duplicated during S phase. Replication timing 35 programs are predictive of genomic features and activity, and considered to be functional 36 readouts of chromatin organization. Although replication timing programs have been described 37 for yeast and animal systems, much less is known about the temporal regulation of plant DNA 38 replication or its relationship to genome sequence and chromatin structure. We used the 39 thymidine analog, 5-ethynyl-2'-deoxyuridine, in combination with flow sorting and Repli-Seq to 40 describe, at high-resolution, the genome-wide replication timing program for Arabidopsis 41 thaliana Col-0 suspension cells. We identified genomic regions that replicate predominantly 42 during early, mid and late S phase, and correlated these regions with genomic features and with 43 data for chromatin state, accessibility and long-distance interaction.

show abstract

Section: Arabidopsis Cell Culture and Nuclei Isolation 652mentioning

confidence: 99%

“…Events were triggered on forward-angle light scatter 685 (FSC), and data were collected using 90° side scatter (SSC) and 460/50 nm and 530/40 nm 686 bandpass filters (Bass et al, 2014;Wear et al, 2016). Plots of SSC vs. 460/50 nm (DAPI) were 687 used to set analysis and sorting gates that excluded cellular debris.…”

Section: Flow Cytometry and Sorting 682mentioning

confidence: 99%

Genome-Wide Analysis of the Arabidopsis Replication Timing Program

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…The fixed, frozen roots described above were ground in cell lysis buffer (CLB from Wear et al, 2016) supplemented with a "Complete Mini" protease inhibitor cocktail tablet (Roche) in a small commercial food processor (Cuisinart Mini-Prep Processor, model DLC-1SS) at 4°C. The resulting homogenate was filtered and centrifuged as previously described (Bass et al, 2015;Wear et al, 2016).…”

Section: Nuclei Isolationmentioning

confidence: 99%

“…The resulting homogenate was filtered and centrifuged as previously described (Bass et al, 2015;Wear et al, 2016). Isolated nuclei were washed in modified CLB buffer (CLB without EDTA or b-mercaptoethanol), and the incorporated EdU was conjugated to AF-488 using a Click-iT EdU Alexa Fluor 488 imaging kit .…”

Section: Nuclei Isolationmentioning

confidence: 99%

“…Events were triggered on forwardangle light scatter and data collected using 90°side scatter and 460/50-nm and 530/40-nm band-pass filters (Bass et al, , 2015Wear et al, 2016). Nuclei prepared from the terminal 1-mm root segments were sorted into 13 NaCl-Tris-EDTA (STE) buffer, pH 7.5, using substage gates to collect populations of EdU/AF-488-labeled nuclei with DNA contents in three defined gates between 2C and 4C, corresponding to early, mid, and late S phase ( Figure 1C).…”

Section: Flow Cytometry and Sortingmentioning

confidence: 99%

See 1 more Smart Citation

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips

Wear

Song²,

Zynda³

et al. 2017

Plant Cell

Self Cite

View full text Add to dashboard Cite

All plants and animals must replicate their DNA, using a regulated process to ensure that their genomes are completely and accurately replicated. DNA replication timing programs have been extensively studied in yeast and animal systems, but much less is known about the replication programs of plants. We report a novel adaptation of the "Repli-seq" assay for use in intact root tips of maize (Zea mays) that includes several different cell lineages and present whole-genome replication timing profiles from cells in early, mid, and late S phase of the mitotic cell cycle. Maize root tips have a complex replication timing program, including regions of distinct early, mid, and late S replication that each constitute between 20 and 24% of the genome, as well as other loci corresponding to ;32% of the genome that exhibit replication activity in two different time windows. Analyses of genomic, transcriptional, and chromatin features of the euchromatic portion of the maize genome provide evidence for a gradient of early replicating, open chromatin that transitions gradually to less open and less transcriptionally active chromatin replicating in mid S phase. Our genomic level analysis also demonstrated that the centromere core replicates in mid S, before heavily compacted classical heterochromatin, including pericentromeres and knobs, which replicate during late S phase.

show abstract

A Protocol for Genome-Wide Analysis of DNA Replication Timing in Intact Root Tips

Mickelson-Young

Wear

Song³

et al. 2021

Methods in Molecular Biology

Self Cite

View full text Add to dashboard Cite

DNA replication during S phase in eukaryotes is a highly-regulated process that ensures the accurate transmission of genetic material to daughter cells during cell division. Replication follows a well-defined temporal program, which has been studied extensively in humans, Drosophila, and yeast, where it is clear that the replication process is both temporally and spatially ordered. The replication timing (RT) program is increasingly considered to be a functional read out of genomic features and chromatin organization. Although there is increasing evidence that plants display important differences in their DNA replication process compared to animals, RT programs in plants have not been extensively studied. To address this deficiency, we 2 developed an improved protocol for the genome-wide RT analysis by sequencing newly replicated DNA ("Repli-seq") and applied it to the characterization of RT in maize root tips. Our protocol uses 5-ethynyl-2'-deoxyuridine (EdU) to label replicating DNA in vivo in intact roots.Our protocol also eliminates the need for synchronization and frequently associated chemical perturbations as well as the need for cell cultures, which can accumulate genetic and epigenetic differences over time. EdU can be fluorescently labeled under mild conditions and does not degrade sub-nuclear structure, allowing for the differentiation of labeled and unlabeled nuclei by flow sorting, effectively eliminating contamination issues that can result from sorting on DNA content alone. We also developed an analysis pipeline for analyzing and classifying regions of replication and present it in a point-and-click application called Repliscan that eliminates the need for command line programming.

show abstract

Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry

Cited by 14 publications

References 19 publications

Genome-Wide Analysis of the Arabidopsis Replication Timing Program

Genome-Wide Analysis of the Arabidopsis Replication Timing Program

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips

A Protocol for Genome-Wide Analysis of DNA Replication Timing in Intact Root Tips

Contact Info

Product

Resources

About