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Status |
Public on Jul 12, 2018 |
Title |
CD4_stimulated_FOSL_ChIP-seq_rep |
Sample type |
SRA |
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Source name |
blood
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Organism |
Homo sapiens |
Characteristics |
tissue: blood cell type: CD4+ T cells treatment: stimulated fixation: fixed antibody: anti-FOSL1 antibody (Santa Cruz Biotechnoloy, catalog# sc-183, lot# 12314)
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Treatment protocol |
The resting CD4+ T cells were activated using anti-CD3/CD28 beads (Invitrogen Dynabeads Human T-Activator CD3/CD28, Cat# 11131D) for 16 hours at 37°C.
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Growth protocol |
Human CD4+ T cells were purified from human blood using human CD4 T cell enrichment Kit from Stemcell Technologies (cat# 19052) according to the manufactures manual. The cells were used directly as resting T cells or activated using anti-CD3/CD28 beads (Invitrogen Dynabeads Human T-Activator CD3/CD28, Cat# 11131D) for 16 hours at 37°C.
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Extracted molecule |
genomic DNA |
Extraction protocol |
The anti-FOSL1 antibody (Santa Cruz Biotechnoloy, Inc. The Cat# is sc-183)-immunoprecipetated chromatin on beads were added with 100ul EB buffer, 3 ul of 10% SDS and 5 ul of 20 mg/ml proteinase K. Incubate at 65C overnight. Extract 1x with 200 ul of phenol/chloroform (PCI) for DNA. Add 1 ul of 20 mg/ml glycogen, final 0.2M NaCl and final 70% Ethanol to precipitate. Incubate at -80°C for 1 hour. Spin at max speed for 30 min. Wash pellet once with 1 ml 70% ethanol, then spin for 10 min. at 4°C. Air dry and resuspend ChIP DNA in 40 ul 1xTE. ChIP DNA is subjected to DN Aend repair using the Epicentre DNA END-Repair kit (Cat. No. ER0720, Epicentre Biotechnologies) according to the manufacturer instruction. Repair DNA was cleaned with the MinElute column using MinElute Reaction Cleanup protocol (Qiagen, pgs 28-29 of MinElute Handbook) and eluted with 40ul EB. Add 5 ul 10x NEB Buffer #2. 1 ul 10 mM dATP and 3 ul 5 U/ul Klenow Fragment (3´→5´ exo–). Incubate for 30 min at 37°C. Purify the reaction again as above and elute the DNA in 22 ul of EB. Add 3 μl 10x T4 DNA ligase buffer, 2 μl adapter (15uM, annealed from primer: multiplexing adapter-top with multiplexing adapter-bottom that is the same as Illumina’s InPE adaptor), mix then add 3 μl T4 DNA ligase (400 units/μl). Incubate for 30 min at room temperature. Add 6 ul H2O and load 18ul per lane on E-gel. Run 10 min. Gel purify the fragments from 200-400 bp. Elute DNA in 22ul EB. Add 25 μl of master mix (HF fusion), 1 μl of 10uM Multiplexing PCR Primer 1.0, 1 μl of 10uM one of the indexed primer2: PCR Primer2- IndexM1-64, or PCR Primer2- IndexM2-64, till PCR Primer2- IndexM12-64 (M stands for Modified, -64 means it is 64bp long). PCR Conditions: 98°C for 30 sec. 17 cycles of 98°C, 10”; 65°C, 30”; 72°C, 30”, 72°C for 5 min. 4°C hold. Gel purify the 250-450 bp fragments for sequencing.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Basecalls performed using CASAVA Reads were aligned to hg18 genome using Bowtie2 Redudant reads or PETs were removed from further analysis For Capture-C datasets, only PETs with bait at promoter of IL2RA were reteined. Reads from replicates were combined. Normalized read count mean value in 21 running window (500 bp) were calucated. Normalized read count were calculated and uploaded to UCSC genome browser. Genome_build: hg18 Supplementary_files_format_and_content: HiC/Capture-C Interaction file: A is the first anchor and B is the second anchor, each row is one interaction event detected by paired-end tag (PET); bedgraph file: First 3 columns indicate genomic coordinate, Column 4 indicates RPBM value.
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Submission date |
May 15, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Keji Zhao |
Organization name |
NHLBI,NIH
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Department |
Systems 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 |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
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Platform ID |
GPL16791 |
Series (1) |
GSE87254 |
Trac-looping measures genome structure and chromatin accessibility |
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Relations |
BioSample |
SAMN07109670 |
SRA |
SRX2821739 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2627260_GB0072_mapq10_noDup_RPBM.bedgraph.gz |
41.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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