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| Status |
Public on Sep 13, 2022 |
| Title |
Stage 8 Pou5f3.3 V5 |
| Sample type |
SRA |
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| Source name |
Whole embryos
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| Organism |
Xenopus laevis |
| Characteristics |
tissue: Whole embryos
strain: Xla.NXR-WTNXR
genotype: WT
developmental stage: NF8
treatment: Injected with 400 pg V5-pou5f3.3 mRNA
antibody: Invitrogen #R960-25
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| Treatment protocol |
For transcription factor CUT&RUN, WT embryos were injected at Stage 1 with 400 pg of N-terminal V5 tagged pou5f3.3.L or sox3.S mRNA.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT&RUN procedure was based on Hainer et al Cell 2019 and Skene et. al. Nature Protocols 2019. 12 embryos per sample were collected, devitellinized in 1mg/mL pronase, dissolved in MR/3, and cells were dissociated in 1 mL of NP2.0 buffer (Briggs et al Science 2018) with gentle agitation. Dissociated cells were lysed in NE buffer (20mM HEPES-KOH, pH 7.9, 10mM KCl, 500M spermidine, 0.1% Triton X-100, 20% glycerol) with gentle pipetting with a clipped P1000, and the lysate was centrifuged at 600xg in 4C for 3 min. The free nuclei were then bound to 300 L of activated concanavalin A beads at RT for 10mins. Nuclei were blocked for 5 min at RT then incubated in 1:100 dilution of primary antibody for 2 hr at 4C, washed, incubated in a 1:200 dilution of pA/G MNase for 1 hr at 4C, and washed again. The bound MNase was activated with 2 mM CaCl2 and allowed to digest for 30 mins, then stopped using 2x STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 g/mL RNase A, 40 g/mL glycogen). Nuclei were incubated at 37C for 20 min followed by centrifuging for 5 min at 16,000xg, drawing off the DNA fragments with the supernatant. The extracted fragments were treated with SDS and proteinase K at 70C for 10 min followed by phenol chloroform extraction. Purified DNA was resuspended in 50 L of water and verified by Qubit dsDNA high sensitivity and Fragment Analyzer.
Sequencing libraries were built using the NEB Next Ultra II library build kit (E7645S) and size selected on an agarose gel to 150-600 bp. Libraries were multiplexed and sequenced paired-end at the Health Sciences Sequencing Core at Children’s Hospital of Pittsburgh.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
s8_pou_v10.1.bw
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| Data processing |
Reads were mapped to the X. laevis v10.1 genome using bowtie2 2.4.2 (--no-mixed --no-discordant), discarding reads on the mitochondrial chromosome. High quality (MAPQ>=30) were retained for analysis, allowing at most 1 fragment per unique coordinate
Paired reads were joined and converted to bigWigs normalized to total number of retained fragments per million, using bedtools v2.30.0 genomecov and wigToBigWig
Assembly: X. laevis v10.1
Supplementary files format and content: bigWig files of coverage pooled across replicates
Library strategy: CUT&RUN
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| Submission date |
Jun 27, 2022 |
| Last update date |
Sep 21, 2023 |
| Contact name |
Miler T Lee |
| E-mail(s) |
miler@pitt.edu
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| Organization name |
University of Pittsburgh
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| Department |
Biological Sciences
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| Street address |
4249 Fifth Ave
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15260 |
| Country |
USA |
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| Platform ID |
GPL21248 |
| Series (2) |
| GSE207025 |
Hybridization led to a rewired pluripotency network in Xenopus laevis [CUT&RUN] |
| GSE207027 |
Hybridization led to a rewired pluripotency network in the allotetraploid Xenopus laevis |
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| Relations |
| BioSample |
SAMN29378270 |
| SRA |
SRX15911795 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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