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| Status |
Public on Mar 19, 2024 |
| Title |
neural crest, Cut and Run, Pax7_3 |
| Sample type |
SRA |
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| Source name |
cranial microdissection
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| Organism |
Gallus gallus |
| Characteristics |
tissue: cranial microdissection
cell type: neural crest
genotype: wt
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| Treatment protocol |
Electroporated embryos were manually dissected, cells were dissociated with 20 strokes using pestle A in isotonic nuclei extraction buffer (NEB: 0.5% NP40, 0.25% Triton X, 10 mM Tris-HCl (pH 7.5), 3 mM CaCl2, 0.25 M sucrose, 1mM DTT, 0.2 mM PMSF, 1X Proteinase inhibitor (PI) ) in a glass homogeniser and cross-linked using 1% formaldehyde at room temperature for 10 min and snap-frozen.
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| Growth protocol |
Fertilised wild-type chicken eggs were obtained from Henry Stewart & Co (Norfolk), staged according to Hamburger and Hamilton (1951) (24). All experiments were performed on chicken embryos younger. than 12 days of development, and as such were not regulated by the Animals (Scientific Procedures) Act 1986.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Snap-frozen embryos were quenched with 125 mM of glycine for 5min. Cross-linker was washed-out by 3x pellet washes with 1x PBS (with 1X PI, 1 mM DTT and 0.2 mM PMSF) centrifuging at 2000 x g for 4min at 4C. Pellets were re-suspended in NEB. Cell nuclei were expulsed with 20 strokes using pestle B in a glass homogeniser, pelleted and washed with 1 xPBS (with 1X PI, 1 mM DTT and 0.2 mM PMSF). Nuclei were lysed in SDS lysis buffer (0.7% SDS, 10mM EDTA, 50 mM Tris-HCl (pH 7.5), 1x PI). Cross-linked chromatin was sonicated at 12A 10x (10s ON 30s OFF) followed by 8A 4x (30s ON 30s OFF). Sheared chromatin samples were pre-cleared in pre-blocked Protein G beads (Dynabeads Protein G, Life Technologies) for 1 hour at 4C. 1/20 of biotinChIP was collected as an input fraction and stored at -80C. Pre-cleared chromatin samples were incubated on pre-blocked streptavidin beads (Dynabeads M-280 streptavidin beads, Life Technologies) o/n at 4C. Beads were washed with SDS Wash Buffer (2% SDS, 10mM Tris-HCl (pH 7.5), 1 mM EDTA) at room temperature, followed by 4x RIPA washes (50 mM Hepes-KOH (pH 8.0), 500 mM LiCl, 1mM EDTA, 1% NP40, 0.7% Na-Deoxycholate, 1x PI) and 1x Na-Cl TE wash (1x TE, 50mM NaCl) at 4C. Chromatin was eluted from the beads with SDS ChIP elution buffer (50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% SDS). Cross-linking was reversed o/n at 70C in the thermomixer at 1300 rpm. Cellular RNA was digested with RNAse A (0.2 g/ml) at 37C for 1 hour, and cellular protein with Proteinase K (0.4 mg/ml) at 55C for 2 hours. Chromatin samples were separated from the streptavidin beads. Input and ChIP DNA was extracted using a standard phenol-chloroform extraction method.
Libraries were prepared using MicropPlex Library Preparation kit (Diagenode) and sequenced using NextSeq 500/550 High Output Kit v2 (75 cycles) on NextSeq 500 sequencing system.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Pax7 CuT_and_Run
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| Data processing |
Reads were mapped using Bowtie -m 2 (v.1.0.0) to galGal6. Read counts were obtained using subread (v.1.4.5) FeatureCounts
Peak calling was performed using MACS2, de novo motif search was performed using Homer (v.4.7)
Assembly: galGal6
Supplementary files format and content: Bigwig files (bw)
Supplementary files format and content: Peaks (bed)
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| Submission date |
Mar 13, 2024 |
| Last update date |
Mar 19, 2024 |
| Contact name |
Tatjana Sauka-Spengler |
| E-mail(s) |
tatjana.sauka-spengler@imm.ox.ac.uk
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| Organization name |
MRC Weatherall Institute of Molecular Medicine
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| Department |
University of Oxford
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| Lab |
Sauka-Spengler lab
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| Street address |
Radcliffe Department of Medicine
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| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
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| Platform ID |
GPL19005 |
| Series (1) |
| GSE261486 |
Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue - specific transcription factors |
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| Relations |
| BioSample |
SAMN40436870 |
| SRA |
SRX23938804 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8145246_smoth_RW-Pax7-CutNRun-HH6.bw |
51.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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