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| Status |
Public on Apr 25, 2023 |
| Title |
Hi-TrAC_cas9_control_rep1 |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
cell type: K562
genotype: CRISPR/Cas9 control
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| Treatment protocol |
Fix cells for Hi-TrAC with 1% Formaldehyde in culture medium at room temperature for 10 minutes. Wash cells twice with ice-cold PBS, then resuspend with 100 µL reaction buffer (50mM Tris-acetate, pH 7.5, 150mM potassium acetate, 10mM magnesium acetate, 4mM spermidine, 0.5% NP-40). Incubate at 37 ℃ for 4 hours. To extract RNA for RNA-seq, add 700 µL QIAzol to 5,000 cells. Cells for ChIP-seq were fixed with 1% Formaldehyde in culture medium at room temperature for 10 minutes. Wash cells with ice-cold PBS twice. Then perform sonication to break chromatins. Sheared chromatin was immunoprecipitated with anti-H3K27ac, anti-H3K4me1and anti-H3K4me3 antibodies. After washing, perform Portease K digestion and reverse cross-linking at 65 ℃ overnight.
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| Growth protocol |
Mouse naïve CD4 T cells were purified from lymph nodes of wildtype or MLL4 knock-out mice. Cells were then activated and cultured in Th17 cells differentiation condition. CTCF-AID and active sub-TAD boundary deletion K562 cells were generated using the CRISPR/Cas9 system. The targeting sequences were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector (Addgene, #62988). For CTCF-AID cells, K562 cells were co-transfected with CRISPR and donor plasmids, which contain Hygromycin selection marker. After 24 hours, cells were treated with 2 g/mL Puromycin and 200 g/mL Hygromycin for 48 hours to remove non-transfected cells. Surviving cells were selected further by treating with 200 g/mL Hygromycin for two weeks. The survived cells were sorted into 96-well plate with one cell per well and cultured for two to three weeks, then genotyped by PCR, western blotting, and sequencing. For generating active sub-TAD boundary deletion cells, K562 cells were transfected with CRISPR plasmids. After 24 hours, cells were treated with 2 g/mL Puromycin for 48 hours to remove non-transfected cells. Survived cells were sorted into 96-well plate with one cell per well, and further cultured for two to three weeks. Cell clones were genotyped by PCR and sequencing.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For Hi-TrAC, genomic DNA was purified by Phenol-Chloroform extraction. For RNA-seq, RNA was extracted with QIAzol lysis reagent (QIAGEN) and RNeasy mini kit (QIAGEN). For ChIP-seq, genomic DNA was purified with QIAGEN MinElute Reaction Cleanup Kit.
For Hi-TrAC, repair gaps of purified genomic DNA with T4 DNA polymerase. Remove free bridging linker using AMPure XP beads. Then, digest purified DNA with MluCI and NlaIII restriction enzymes. Enrich biotin-labeled DNA fragments with streptavidin beads. Perform adapter ligation on beads. After washing the beads, amplify the library by PCR with illumina multiplexing indexed primers. For RNA-seq, libraries were constructed following Smart-seq2 protocol (Picelli et al., 2014). For ChIP-seq, DNA was purified, then end-repaired using End-It DNA End-Repair kit (Epicentre). A-tailing was performed with Klenow Fragment (3'->5' exo-) at the presence of dATP. Universal adapters were then ligated. The libraries were then amplified by PCR using indexed primers.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Mouse sequencing data were processed to the reference genome mm10, and human data were processed to hg38. Raw reads of RNA-seq data were mapped by STAR (v2.7.3a) (Dobin et al., 2013) and quantified by cufflinks. Raw reads of ChIP-seq data were mapped by Bowtie2 (Langmead and Salzberg, 2012). Mapped unique paired-end reads with MAPQ 10 were used for the following analysis. Raw reads of Hi-TrAC data were processed by tracPre2.py in cLoops2 package into unique nonredundant interacting PETs.
Assembly: hg38, mm10
Supplementary files format and content: BEDPE files for Hi-TrAC and ChIP-seq were provided containg processed high-quality and non-redundant paired-end tags. TXT file for gene expression quantified in FPKM is also provided.
Library strategy: Hi-TrAC
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| Submission date |
Feb 15, 2023 |
| Last update date |
Apr 25, 2023 |
| Contact name |
Yaqiang Cao |
| E-mail(s) |
caoyaqiang0410@gmail.com
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| Organization name |
NHLBI
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| Department |
System Biology Center
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| Lab |
Laboratory of Epigenome Biology
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| Street address |
9000 Rockville Pike
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| City |
Bethesda |
| State/province |
Maryland |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE208085 |
Hi-TrAC Detects Active Sub-TADs and Reveals Internal Organizations of Super-Enhancers |
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| Relations |
| BioSample |
SAMN33315868 |
| SRA |
SRX19383403 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7047500_Hi-TrAC_cas9_control_rep1_unique.bedpe.gz |
334.6 Mb |
(ftp)(http) |
BEDPE |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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