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| Status |
Public on Dec 31, 2022 |
| Title |
dCas9-MRE11 rep1 |
| Sample type |
SRA |
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| Source name |
human embryonic stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: embryonic stem cells
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| Growth protocol |
hESCs were cultured in matrigel-coated plates in the Essential 8TM hESC medium (Thermo, catalog. no. A1517001). The medium was changed daily. hESCs were treated with 0.48 mM EDTA for dissociation and then passaged onto new Matrigel -coated plates every 4–5 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
A hyperactive pA-Tn5 transposon for the illumina platform was first generated. hESCWT, hESCiCas9 and hESCidCas9 were treated with 1 µg/ml doxycycline for 12 hours. Cells were harvested and washed twice. Then the nuclei were isolated by ice-cold NE1 buffer and re-suspended in wash buffer. 10 μl of activated concanavalin A beads were added per sample and incubated at room temperature for 10 minutes. Primary antibody (1:50 dilution of anti-IgG, anti-FLAG (Cas9), anti-HA (dCas9), anti-MRE11) incubation was performed on a rotator at 4°C overnight. To increase the number of protein A binding sites, a secondary antibody (such as guinea pig anti-Rabbit IgG antibody for a rabbit primary antibody, and rabbit anti-mouse IgG for a mouse primary antibody) was diluted 1:100 in wash buffer and incubated at RT for 1h. After removing unbound antibodies, cells were mixed with 1:100 dilution of pA-Tn5 (0.04 µM) and incubated at room temperature for 1 hour on rotator. Next, cells were re-suspended in 300 µl tagmentation buffer and incubated at 37 °C for 1 h. To stop tagmentation, 10 µl 0.5 M EDTA, 3µl 10% SDS and 2.5 µl of 20 mg/mL Proteinase K was added to sample, which was incubated overnight at 37 °C. DNA was extracted by PCI and dissolved in 30 μl TE buffer.
To amplify libraries, we added P5 primer, P7 primer (Vazyme, catalog. no. TD202), TruePrep Amplify Enzyme (Vazyme catalog. no. TD601) and performed PCR with the conditions below: 72°C for 3 minutes; 98°C for 30 seconds; 18 cycles of 98°C for 15 seconds, 63°C for 15 seconds; 72°C for 5 minutes. Post-PCR clean-up was performed by adding 1.2 volume of DNA clean beads (Vazyme, catalog. no. N411) and libraries were eluted in 20 μl nuclease-free water.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
d-MRE-R1
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| Data processing |
Library strategy: CUT&Tag
Raw data were first trimmed and filtered by TrimGalore with default parameter; then qualified read pairs were aligned to reference genome by Bowtie2 (release 2.4.1); after that biological and optical duplicates were removed with Sambamba (release 0.7.1); last we used MACS2 (release 2.2.7.1; parameters: -q 0.05 -g hs -f BAMPE) to call peaks. All Cut-Tag peaks were filtered with 5% FDR threshold.
We abolished the filtering step in which BLENDER (https://github.com/staciawyman/blender) compares the similarity known gRNA sequence and putative cut sites to discard some candidates. In our code, the cut sites with highest sequencing signal were retained and provided for downstream analyses and experimental validation.
Genome_build: hg38
Supplementary_files_format_and_content: BED format file (Cas9 and MRE11 CutTag peaks and putative cut sites of iCas9)
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| Submission date |
Nov 17, 2020 |
| Last update date |
Dec 31, 2022 |
| Contact name |
Denghui Chen |
| E-mail(s) |
chendh39@mail.sysu.edu.cn
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| Organization name |
Sun Yat-sen University
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| Department |
Zhongshan School of Medicine
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| Street address |
No. 74 Zhongshan 2nd Road
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| City |
Guangzhou City |
| ZIP/Postal code |
510080 |
| Country |
China |
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| Platform ID |
GPL18460 |
| Series (2) |
| GSE161633 |
Cas9 interactions with host RNAs and transposable elements impair neurodevelopment (CUT&Tag) |
| GSE161635 |
Cas9 interactions with host RNAs and transposable elements impair neurodevelopment |
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| Relations |
| BioSample |
SAMN16817742 |
| SRA |
SRX9521148 |
| 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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