Name: Spaltning under mål og frigjøring ved hjelp av nukleaseanalyse av FACS-sorterte musesatellittceller
Uploaded: 2023-07-07T14:20:00
Description: Watch this Scientific Journal Video about Cleavage Under Targets and Release Using Nuclease Analysis of FACS-Sorted Mouse Satellite Cells at JoVE.com

cleavage under targets and release using nuclease analysis of facs-sorted mouse satellite cells

Satellittcelleisolering fra musemuskler og flowcytometrianalyse

Skeletal striated muscles represent on average 40% of the weight of the total human body1. Muscle fibers exhibit a remarkable capacity for regeneration upon injury, which is described by the fusion of newly formed myocytes and the generation of new myofibers that replace the damaged ones2. In 1961, Alexander Mauro reported a population of mononuclear cells that he termed as satellite cells3. These stem cells express the transcription factor paired box 7 (PAX7), and are located between the basal lamina and the sarcolemma of muscle fibers4. They were reported to express the cluster of differentiation 34 (CD34; a hematopoietic, endothelial progenitor and mesenchymal stem cell marker), integrin alpha 7 (ITGA7; a smooth, cardiac and skeletal muscle marker), as well as the C-X-C chemokine receptor type 4 (CXCR4; a lymphocyte, hematopoietic, and satellite cell marker)5. In basal conditions, satellite cells reside in a particular microenvironment that keeps them in a quiescent state6. Upon muscle damage, they become activated, proliferate, and undergo myogenesis7. However, contributing only to a minor fraction of the total number of muscle cells, their genome-wide analyses are particularly challenging, especially under physiological settings (&#60;1% of total cells).
Various methods for chromatin isolation from satellite cells have been described, which involve chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) or cleavage under targets and tagmentation (CUT&#38;Tag) experiments. Nevertheless, these two techniques present some significant limitations that remain unchallenged. Indeed, ChIP-seq requires a high amount of starting material to generate enough chromatin, a large proportion of which is lost during the sonication step. CUT&#38;Tag is more appropriate for low cell number, but generates more off-target cleavage sites than ChIP-seq due to the Tn5 transposase activity. In addition, since this enzyme has a high affinity for open-chromatin regions, the CUT&#38;Tag approach might be preferentially used for analyzing histone modifications or transcription factors associated with actively transcribed regions of the genome, instead of silenced heterochromatin8,9.
Presented here is a detailed protocol that allows the isolation of mouse limb muscle satellite cells by FACS for cleavage under targets and release using nuclease (CUT&#38;RUN)10,11 analysis. The various steps involve the mechanical disruption of tissue, cell sorting, and nuclei isolation. The method&#39;s efficiency, regarding the preparation of a viable cell suspension, was demonstrated by performing CUT&#38;RUN analysis for covalent histone modifications and transcription factors. The quality of isolated cells makes the described method particularly attractive for preparing chromatin that captures the native genomic occupancy state faithfully, and is likely to be suitable for capturing the chromosome conformation in combination with high-throughput sequencing at specific loci (4C-seq) or at genome-wide levels (Hi-C).

Forfatter Spotlight: Cistromeanalyse i musemuskelstamceller 

En effektiv protokoll for CUT&amp;RUN-analyse av FACS-isolerte musesatellittceller

Since more than 20 years, our lab is interested in the pathophysiological role of nuclear receptors. To decipher their in vivo function, we engineered mouse model allowing the spatial and temporal control of their expression. In parallel, we developed cutting-edge techniques to investigate their molecular function in the mouse tissues.Our scientific interest recently led us to determine secosteroid and steroid receptor signaling in various tissues, including skeletal muscle. We have recently demonstrated that the androgen receptor in myofibers that differentiate cells of the skeletal muscle is instrumental in their contractile and metabolic functions. Our recent findings deliver a deeper understanding of skeletal muscle pathophysiological dynamics that is crucial to develop effective treatments for muscle-associated disorders.We developed this protocol to be able to perform cistrome analysis on muscle stem cells in vivo. All former protocols used in vitro modals, which completely dissociates the investigation from a whole organism context. In addition to being low-cost and time-efficient, this protocol provides an effective experimental setting to study transcription factor recruitment and chromatin landscape in skeletal muscle progenitor cells.Chromatin prepared by this protocol has provided the first genome-wide analysis of AR cistrome in satellite cells, and will facilitate future studies on gene regulation. Among many, our future aim would be to extend these investigations onto other oxosteroid receptors and explore their specific co-regulators in the various cell types involved in the regeneration process in skeletal muscle.

Watch this Scientific Journal Video about Cleavage Under Targets and Release Using Nuclease Analysis of FACS-Sorted Mouse Satellite Cells at JoVE.com

Spaltning under mål og frigjøring ved hjelp av nukleaseanalyse av FACS-sorterte musesatellittceller

Sentrifuger de FACS-isolerte satellittcellene og kast supernatanten. Vask cellene med en milliliter PBS, sentrifuge, og inkuber dem i en milliliter kald kjernefysisk ekstraksjonsbuffer på is i 20 minutter. Tilsett 20 mikroliter Concanavalin A belagte magnetiske perler i et 1,5 milliliter rør som inneholder 850 mikroliter kaldbindingsbuffer.Bruk deretter et magnetisk stativ, vask perlene to ganger med en milliliter kaldbindingsbuffer og la perlene samle seg på siden av røret på stativet i fem minutter før du forsiktig resuspenderer dem i 300 mikroliter kaldbindingsbuffer. Deretter sentrifugerer du kjernene og resuspenderer dem forsiktig i 600 mikroliter kjernefysisk ekstraksjonsbuffer. Bland deretter 600 mikroliter ekstraherte kjerner med 300 mikroliter Concanavalin A-perleoppslemming og inkuber ved fire grader Celsius i 10 minutter.Etter å ha fjernet supernatanten ved hjelp av et magnetisk stativ, resuspender de perlebundne kjernene med en milliliter kaldblokkerende buffer. Fjern igjen supernatanten og vask de perlebundne kjernene to ganger med en milliliter kaldvaskbuffer. Separer supernatanten, resuspender forsiktig kjerneperlekompleksene med et spesifikt primært antistoff og inkuber ved fire grader Celsius over natten med mild omrøring.Neste dag, fjern supernatanten og vask de perlebundne kjernene før du resuspenderer i 100 mikroliter kaldvaskbuffer. Tilsett 100 mikroliter fortynnet protein A-mikrokokknuklease til 100 mikroliter perlebundne kjerner og inkuber ved fire grader Celsius i en time med omrøring. Etter inkubering, fjern supernatanten og vask de perlebundne kjernene to ganger før de resuspenderes i 150 mikroliter kaldvaskbuffer.Deretter tilsettes tre mikroliter 100 millimolært kalsiumklorid, blandes raskt ved å flikke og inkubere på is i 30 minutter. Stopp reaksjonen ved å tilsette 150 mikroliter stoppbuffer og inkuber ved 37 grader Celsius i 20 minutter. Sentrifuger prøvene og overfør supernatanten til et nytt mikrosentrifugerør, og kast pelleten og perlene.Deretter tilsettes tre mikroliter natriumdodecylsulfat og 2,5 mikroliter 20 milligram per milliliter proteinase K.Mix ved inversjon og inkuberes i 10 minutter ved 70 grader Celsius. Deretter tilsettes 300 mikroliter fenol: kloroform: isoamylalkohol og hvirvel før overføring til to milliliter faselåsrør. Sentrifuge og tilsett 300 mikroliter kloroform til samme rør.Sentrifuge igjen og samle supernatanten i et 1,5 milliliter rør. Deretter tilsettes en mikroliter glykogen, deretter 750 mikroliter 100% etanol, og la DNA utfelle over natten ved minus 20 grader Celsius. Neste dag, pellet DNA ved sentrifugering, vask deretter pelleten med en milliliter 100% etanol og sentrifuge to ganger før du fjerner gjenværende etanol med en pipette.Lufttørk pelleten i fem minutter og resuspender den i 25 mikroliter tris-EDTA-buffer. H3KME2 og H3K27AC ble anriket rundt TSS av PAC7, ITGA7, LAM2, CXCR4 og VCAM1. Imidlertid ble H3K4ME2 og H3K27AC ikke beriket på de av ITGAM, PTPRC, PECAM, CKM eller MHY3.Nesten ingen signal ble oppnådd med IgG-prøven. HOMER kjent motivanalyse, AR-topper og satellittceller og prosent målrettede regioner vises. Søkeranalyse avduket nesten 500 topper med en toppscore høyere enn 50 med mer enn 200 av dem med en poengsum høyere enn 100.

Research

JoVE Journal

Developmental Biology

Cleavage Under Targets and Release Using Nuclease Analysis of FACS-Sorted Mouse Satellite Cells

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...