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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 17, 2021 |
| Title |
4C_Muscle_Myh3 |
| Sample type |
SRA |
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| Source name |
Muscle
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| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl6N
tissue: Gastrocnemius+Tibialis+Quadriceps
age: 8 weeks
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| Treatment protocol |
Circular Chromosome Conformation Capture (4C) technique was performed as described (Matelot and Noordermeer, 2016 Meth Mol Biol). Cross-linked chromatin was digested with DpnII, ligated under diluted conditions, cross links were reversed and DNA was further digested by NlaIII and again ligated under diluted conditions.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Muscle nuclei were isolated with hypotonic buffer (25mM Hepes-KOH pH 7.8, 10mM KCL, 1.5mM MgCl2, 0.1% NP40, PIC 1X (complete protease inhibitor Roche), PMSF 1mM). Muscles were sliced with a scissor and then homogenized with an Ultra-Turrax. The solution was transferred in a 15 ml Falcon tube and crosslinked with 2% formaldehyde at room temperature during 10 min. 1,43 ml of cold glycine (1M) was added to quench the formaldehyde for 5 min at +4°C while shaking. The crosslinked nuclei were dounced 10 times with a loose pestle and then centrifuged at 1000 Rcf for 10 min at +4°C. The nuclear pellet was resuspended in 5ml of hypotonic buffer and filtered with 70µm and 40µm cells strainers. The nuclei were pelleted with centrifugation at 1000 rcf for 10 min, snap frozen into liquid nitrogen and stored at -80°C.
PCR amplification using primers with included Tru-seq adapter sequences and indexes.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
4C–seq libraries were constructed according to Matelot and Noordermeer, Meth Mol Biol 2016. 4C-seq libraries were prepared from indicated tissue samples. The primary restriction enzyme used was DpnII (New England Biolabs, R0543M) and the secondary restriction enzyme was NlaIII (New England Biolabs, R0125L). For each viewpoint approximately 1 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina Tru-seq adapters. Illumina adapters (single end reads) were included in the inverse 4C PCR primers used for 4C-seq library preparation. Multiplexed samples were sequenced on the Illumina NextSeq 550 system using 86 bp single-end reads according to the manufacturer’s specifications. 4C-seq reads were sorted, aligned, and translated to restriction fragments using the 4C-seq pipeline of the BBCF HTSstation (David et al., 2014 Plos ONE). Samples were mapped to the ENSEMBL Mouse assembly NCBIM38 (mm10).
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| Data processing |
Library strategy: 4C-seq
De-multiplexing, mapping and 4C-seq were done through HTSstation (David et al, 2014 PLoS ONE).
Reads were mapped to the mm10 (NCBIM38) mouse genome assembly with bowtie version 0.12.7 (Langmead et al, 2009 Genome Biology) with parameters -n 2 --best -strata -m 5.
Mapped reads were translated to restriction fragments using the HTSstation (David et al, 2014 PLoS ONE).
4C figures were made using a running mean algorithm with a window size of eleven fragments (mappable restriction fragments with 0 values included).
Genome_build: mm10
Supplementary_files_format_and_content: Signal/restriction fragment (bw) and smoothed signal/restriction fragment (bedGraph), one per viewpoint, per cell type.
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| Submission date |
Mar 02, 2021 |
| Last update date |
Dec 17, 2021 |
| Contact name |
Matthieu Dos Santos |
| Organization name |
Institut Cochin
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| Street address |
24 rue du Faubourg Saint Jacques
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| City |
Paris |
| State/province |
Paris |
| ZIP/Postal code |
75014 |
| Country |
France |
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| Platform ID |
GPL21626 |
| Series (1) |
| GSE168074 |
A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes |
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| Relations |
| BioSample |
SAMN18108792 |
| SRA |
SRX10207101 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5127879_4C_Muscle_Myh3_segToFrag.bw |
915.7 Kb |
(ftp)(http) |
BW |
| GSM5127879_4C_Muscle_Myh3_segToFrag_smoothed_11FragsPerWin.bedGraph.gz |
2.2 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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