|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Apr 25, 2023 |
Title |
Hi-TrAC_WT_Th17_72h_rep2 |
Sample type |
SRA |
|
|
Source name |
Th17
|
Organism |
Mus musculus |
Characteristics |
strain: mixed C57BL/6 and 129 cell type: CD4+ T cells stimulated for 72 h (horizontal axis) under Th17 condition tissue: Cell Culture genotype: WT DAPI-
|
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.
|
Growth protocol |
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.
|
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.
|
|
|
Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2000 |
|
|
Data processing |
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: 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
|
|
|
Submission date |
Jul 13, 2022 |
Last update date |
Apr 25, 2023 |
Contact name |
Yaqiang Cao |
E-mail(s) |
caoyaqiang0410@gmail.com
|
Organization name |
NHLBI
|
Department |
System Biology Center
|
Lab |
Laboratory of Epigenome Biology
|
Street address |
9000 Rockville Pike
|
City |
Bethesda |
State/province |
Maryland |
ZIP/Postal code |
20892 |
Country |
USA |
|
|
Platform ID |
GPL13112 |
Series (1) |
GSE208085 |
Hi-TrAC Detects Active Sub-TADs and Reveals Internal Organizations of Super-Enhancers |
|
Relations |
BioSample |
SAMN29680370 |
SRA |
SRX16174111 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6336912_Hi-TrAC_WT_Th17_72h_rep2_unique.bedpe.gz |
632.1 Mb |
(ftp)(http) |
BEDPE |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
|
|
|
|
|