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| Status |
Public on Jun 24, 2024 |
| Title |
CUTTag_hESC_control_input |
| Sample type |
SRA |
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| Source name |
Human embryonic stem cells
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: anti-flag antibody (Sigma, Cat# F7425)
cell line: Human embryonic stem cells
genotype: WT
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Human ES cells were cultured in Essential-8 medium (Thermo Fisher, Cat# A1517001) containing Matrigel (Corning, Cat# 354277). Cells were dissociated using Versene Solution (Gibco, Cat# 15040066)
Hyperactive Universal CUT&Tag Assay Kit (Vazyme, Cat# TD904) was used to construct the CUT&Tag libraries of OTX2-overexpressed hESCs according to the manufacturer’s protocol. hESCs that were not transfected with OTX2-overexpression plasmid were used as the negative control. Briefly, 100,000 EGFP-positive OTX2-overexpressed hESCs or control hESCs were washed and resuspended in 100 μL wash buffer before 10 µL of activated Concanavalin A coated magnetic beads were added to each sample and incubated at room temperature (RT) for 10 mins. After that, cells were incubated with 4 µg of anti-flag antibody (Sigma, Cat# F7425) on a rotating platform for 2 hrs at RT. Then, Anti-rabbit IgG antibody (Abcam, Cat# ab6702) was added and incubated at RT for 60 mins. Next, cells were washed three times with Dig-Wash buffer to remove unbound antibodies. After that, pA/G-Tnp incubation was performed on a rotating platform for 1 hr at RT. Similarly, cells were washed three times with Dig-300 buffer to remove unbound pA/G-Tnp protein and resuspended in tagmentation buffer and incubated at 37ºC for 1 hr. Tagmentation was stopped by adding of 10% SDS and incubating at 55 ºC for 10 mins. Then cells were plated on a magnet rack, supernatant was transferred into a new PCR tube and DNA was extracted by using DNA Extract Beads Pro. Finally, libraries were constructed using TruePrep Index Kit V2 for Illumina (Vazyme, Cat# TD202). All CUT&Tag libraries were sequenced using the Illumina NovaSeq 6000 platform with PE150 sequencing strategy.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
CUTTag_hESC_OE_OTX2.cleanPeaks.bed
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| Data processing |
Adaptors were trimmed from raw sequences using Trim Galore (v0.6.6). The trimmed reads were aligned to the human reference genome (hg19) using bowtie2 with parameters: --local --verysensitive --no-mixed --no-discordant --phred33 -I 10 -X 700. PCR duplicates were identified and removed using sambamba (v0.8.2). Unmapped reads were further excluded using samtools (v1.17). Read pairs that were on the same chromosome and fragment length less than 1000 bp were used for the downstream analysis. Peak calling was performed using SEACR (v1.3). The enriched regions in OTX2-overexpressed hESC were called using the wildtype hESC as control track.
Assembly: hg19
Supplementary files format and content: bigWig, OTX2 binding signals; bed, SEACR called peaks
Library strategy: CUT&Tag
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| Submission date |
Jun 20, 2024 |
| Last update date |
Jun 24, 2024 |
| Contact name |
Fan Guo |
| E-mail(s) |
[email protected]
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| Organization name |
Institute of Zoology, Chinese Academy of Sciences
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| Department |
The State Key Laboratory of Stem Cell and Reproductive Biology
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| Lab |
Group of Reproductive Epigenetics
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| Street address |
1 Beichen West Road, Chaoyang District
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| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE224618 |
Divergent originations of parental DNA hydroxymethylation in human preimplantation embryos |
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| Relations |
| BioSample |
SAMN41940670 |
| SRA |
SRX24993271 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8342376_CUTTag_hESC_control_input_RPKM_bin50.bw |
72.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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