High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids

Cerrizuela, Santiago; Kaya, Oğuzhan; Kremer, Lukas P.M.; Sarvari, Andrea; Ellinger, Tobias; Straub, Jannes; Brunken, Jan; Sanz-Morejón, Andrés; Korkmaz, Aylin; Martín-Villalba, Ana

doi:10.1016/j.xpro.2022.101555

Cited by 3 publications

(3 citation statements)

References 12 publications

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“…Applying ptalign to tumor-cell hierarchies, we conducted pseudotime alignment of healthy v-SVZ NSCs with 4 replicates of a GBM patient derived xenograft (PDX, pseudonymously T6) sequenced 5 months post-injection (mpi) by a miniaturized and automated Smart-seq2/3 protocol 27 (SFig.3a-d). This alignment is shown schematically in (Fig.1c).…”

Section: Resultsmentioning

confidence: 99%

“…The genomic DNA (gDNA) from 3 whole-brain sections per group was isolated by QiaAmp DNA microkit (Qiagen 1048145) and the concentrations were measured by Qubit DNA HS kit. The protocol for bisulfite conversion and library preparation was adapted based on the genomic part of the scNMT-seq protocol 12 with the following modifications. The gDNA input was adjusted to be 20 ng per sample.…”

Section: Methodsmentioning

confidence: 99%

“…WGBS libraries were prepared as described in 12 in triplicate from PDX samples. We generated a combined human-mouse reference genome per 32 to map reads from mouse and human cells using Bismark v0.20 33 with Bowtie2 v2.3.5.1 34 .…”

Section: Methodsmentioning

confidence: 99%

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Identification of astrocyte-driven pseudolineages reveals clinical stratification and therapeutic targets in Glioblastoma

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Kaya,

Wüst

et al. 2023

Preprint

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Cancer research has predominantly targeted genetic mutations, while only recently has attention shifted to understanding tumor cell-stages. However, the key organizational principles guiding tumor dynamics towards sustainable growth remained unexplored. By analyzing tumor cell ensembles from individuals with glioblastoma through the lens of the healthy adult stem cell lineage, we identified astrocytes as central to glioblastoma progression. We found dormant tumor cells resembling astrocytes progressing to active and differentiated stages, building tumor pseudolineages that ultimately influence patient survival. These tumor stages align with specific methylomes, offering potential for patient classification. Our study identifies the Wnt antagonist SFRP1 as a missing factor in glioblastoma that plays a crucial role in the transition from quiescence to activation in the healthy lineage. Excitingly, re-introduction of SFRP1 in glioblastoma halts tumor dynamics, enhancing survival in a PDX model. This fresh view on glioblastomas underscores the importance of understanding tumor dynamics and unveils novel therapeutic avenues.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Identification of astrocyte-driven pseudolineages reveals clinical stratification and therapeutic targets in Glioblastoma

Foerster,

Kaya,

Wüst

et al. 2023

Preprint

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Single-cell triple-omics uncovers DNA methylation as key feature of stemness in the healthy and ischemic adult brain

Kremer

Cerrizuela

Shukairi

et al. 2022

Preprint

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Stem cells in the adult brain are specialized astrocytes capable of generating neurons and glial cells. While neural stem cells (NSCs) and common astrocytes have clearly distinct functions, they share highly similar transcriptome profiles. How stemness is molecularly encoded is therefore unclear. Here we use single-cell NMT-seq to simultaneously characterize the transcriptome, DNA methylome and chromatin accessibility of astrocytes and the NSC lineage in the healthy and ischemic brain. Our data reveal distinct methylation profiles associated with either astrocyte or stem cell function. Stemness is conferred by methylation of astrocyte genes and demethylation of neurogenic genes that are expressed only later. Surprisingly, ischemic injury unlocks the stemness-methylome in common astrocytes enabling generation of neuroblasts. Furthermore, we show that oligodendrocytes employ Tet-mediated demethylation to regulate expression of myelin-related genes, many of which are abnormally methylated in multiple sclerosis. Overall, we show that DNA methylation is a promising target for regenerative medicine.

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Technologies and applications of single-cell DNA methylation sequencing

Liu¹,

Wang²,

Gu³

et al. 2023

Theranostics

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High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids

Cited by 3 publications

References 12 publications

Identification of astrocyte-driven pseudolineages reveals clinical stratification and therapeutic targets in Glioblastoma

Identification of astrocyte-driven pseudolineages reveals clinical stratification and therapeutic targets in Glioblastoma

Single-cell triple-omics uncovers DNA methylation as key feature of stemness in the healthy and ischemic adult brain

Technologies and applications of single-cell DNA methylation sequencing

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