Targeted Bisulfite Sequencing Using the SeqCap Epi Enrichment System

Wendt, Jennifer; Rosenbaum, Heidi; Richmond, Todd; Jeddeloh, Jeffrey A.; Burgess, Daniel L.

doi:10.1007/978-1-4939-7481-8_20

Cited by 24 publications

(14 citation statements)

References 10 publications

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“…The 1010 unique genes from these combined lists were selected for enrichment in bisulfite‐seq using a custom SeqCap Epi Enrichment System (Roche, Pleasonton, CA, USA; Table S1). 38 Targeted bisulfite sequencing was performed at the Penn State Huck Institutes of the Life Sciences Genomics Core Facility. Libraries were constructed using the KAPA Hyper Prep kit (Kapa Biosystems, Wilmington, MA).…”

Section: Methodsmentioning

confidence: 99%

Paternal nicotine enhances fear memory, reduces nicotine administration, and alters hippocampal genetic and neural function in offspring

Goldberg

Zeid

Kutlu³

et al. 2019

Addiction Biology

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Nicotine use remains highly prevalent with tobacco and e-cigarette products consumed worldwide. However, increasing evidence of transgenerational epigenetic inheritance suggests that nicotine use may alter behavior and neurobiology in subsequent generations. We tested the effects of chronic paternal nicotine exposure in C57BL6/J mice on fear conditioning in F1 and F2 offspring, as well as conditioned fear extinction and spontaneous recovery, nicotine self-administration, hippocampal cholinergic functioning, RNA expression, and DNA methylation in F1 offspring. Paternal nicotine exposure was associated with enhanced contextual and cued fear conditioning and spontaneous recovery of extinguished fear memories. Further, nicotine reinforcement was reduced in nicotine-sired mice, as assessed in a selfadministration paradigm. These behavioral phenotypes were coupled with altered response to nicotine, upregulated hippocampal nicotinic acetylcholine receptor binding, reduced evoked hippocampal cholinergic currents, and altered methylation and expression of hippocampal genes related to neural development and plasticity. Gene expression analysis suggests multigenerational effects on broader gene networks potentially involved in neuroplasticity and mental disorders. The changes in fear conditioning similarly suggest phenotypes analogous to anxiety disorders similar to post-traumatic stress.

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Section: Methodsmentioning

confidence: 99%

Paternal nicotine enhances fear memory, reduces nicotine administration, and alters hippocampal genetic and neural function in offspring

Goldberg

Zeid

Kutlu³

et al. 2019

Addiction Biology

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“…Sample DNA was then bisulfite converted following the EZ DNA Methylation-Lightning Kit (Zymo Research; CA, USA). Sample DNA was then PCR amplified, hybridized with custom capture probes, and PCR amplified again in accordance with Roche SeqCap Epi system protocols [68]. Sample captured bisulfite-converted DNA sequencing was performed by Genome Quebec on the Illumina HiSeq 2500 following Illumina guidelines.…”

Section: Isolation Of Prefrontal Cortex Dna Capture and Bisulfite Sementioning

confidence: 99%

The Transition from Acute to Chronic Pain: Dynamic Epigenetic Reprogramming of the Mouse Prefrontal Cortex up to One Year Following Nerve Injury

Topham

Grégoire

Kang

et al. 2020

Preprint

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Chronic pain is associated with persistent structural and functional changes throughout the neuroaxis, including in the prefrontal cortex (PFC). The PFC is important in the integration of sensory, cognitive and emotional information and in conditioned pain modulation. We previously reported wide-spread epigenetic reprogramming in the PFC many months following nerve injury in rodents. Epigenetic modifications, including DNA methylation, can drive changes in gene expression without modifying DNA sequences.To date, little is known about epigenetic dysregulation at the onset of acute pain or how it progresses as pain transitions from acute to chronic. We hypothesize that acute pain following injury results in rapid and persistent epigenetic remodelling in the PFC that evolves as pain becomes chronic. We further propose that understanding epigenetic remodelling will provide insights into the mechanisms driving pain-related changes in the brain. Epigenome-wide analysis was performed in the mouse PFC 1 day, 2 weeks, 6 months, and 1 year following peripheral injury using the spared nerve injury (SNI) in mice.SNI resulted in rapid and persistent changes in DNA methylation, with robust differential methylation observed between SNI and sham-operated control mice at all time points.Hundreds of differentially methylated genes were identified, including many with known function in pain. Pathway analysis revealed enrichment in genes related to stimulus response at early time points, immune function at later time points and actin and cytoskeletal regulation throughout the time course. Increased attention to pain chronicity as a factor is recommended for both pain research and management.

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“…These capturing and enrichment steps allow us to obtain a reliable depth of DNA methylation data at the CpG site level. This method has the advantage of selecting the largest difference in DNA methylation levels and the most clinically relevant CpG sites among CpG islands or other genomic regions 22 . However, a more straightforward methylation method, methylation-specific polymerase chain reaction (MS-PCR, MSP), has been developed and used to validate the methylation status 23 .…”

Section: Introductionmentioning

confidence: 99%

Hypermethylation of PDX1, EN2, and MSX1 predicts the prognosis of colorectal cancer

et al. 2022

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Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.

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Targeted Bisulfite Sequencing Using the SeqCap Epi Enrichment System

Cited by 24 publications

References 10 publications

Paternal nicotine enhances fear memory, reduces nicotine administration, and alters hippocampal genetic and neural function in offspring

Paternal nicotine enhances fear memory, reduces nicotine administration, and alters hippocampal genetic and neural function in offspring

The Transition from Acute to Chronic Pain: Dynamic Epigenetic Reprogramming of the Mouse Prefrontal Cortex up to One Year Following Nerve Injury

Hypermethylation of PDX1, EN2, and MSX1 predicts the prognosis of colorectal cancer

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