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| Status |
Public on Jan 20, 2019 |
| Title |
KO SEL_Input |
| Sample type |
SRA |
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| Source name |
Thymocytes
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| Organism |
Mus musculus |
| Characteristics |
cell type: TCR-int CD69-pos thymocyte
strain: C57BL/6
genotype: OT-II CD2-icre HDAC3-cKO
chip antibody: none
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Thymi were harvested, processed for single cell suspension, and stained with antibody for cell sorting by FACS Aria. Sorted samples were cross-linked with 1% formaldehyde (final concentration) for 10 min and quenched with 125 mM glycine for 5 min at room temperature. After washing with TBS, cells were resuspended in cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. Lysates were washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2). After resuspendsion in 200 μL fresh MNase digestion buffer containing a proteinase inhibitor cocktail (Sigma), the lysates were incubated with 2,000 units of MNase (NEB, Cat # M0247S) per 4 x 106 cells at 37 °C for 20 min with continuous mixing in thermal mixer. After adding 200 μL of sonication buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), the lysates were sonicated for 30 min (30 sec on / 30 sec off) using Bioruptor Twin (UCD-400) (Diagenode, Inc.) and centrifuged at 21,130 x g for 10 min. The supernants were collected, and the chromatin content was estimated by the Qubit assay (Invitrogen). For normalization of ChIP efficiency, Drosophila chromatin equivalent to about 5% of total chromatin was added. The chromatin was then incubated with anti-H3K27ac antibody (Abcam, Cat.# ab4729, lot# GR150367) or in-house generated anti-H3K9ac antibody (EDL lot 1) on a rocker overnight. Protein G-magnetic beads (30 μL, Life Technologies) were added, and further incubated for 3 hours. The beads were extensively washed with ChIP buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl2 buffer (10 mM Tris-HCl, pH8.0, 0.25 M LiCl2, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and TE buffer. Bound chromatin was eluted and reverse-crosslinked at 65°C overnight. DNAs were purified using Min-Elute PCR purification kit (Qiagen) after the treatment of RNase A and proteinase K.
ChIP-seq libraries were prepared from about 5 ng ChIP and input DNA using the ThruPLEX® DNA-seq Kit V2 (Rubicon Genomics, Ann Arbor, MI) according to manufacturere protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
ChIP-seq data was analyzed using the HiChIP pipeline (Yan et al., 2014). In a brief, raw reads were mapped to the reference genome (mm10) using BWA (v0.5.9) with default parameters for pair-end reads. Uniquely mapped reads were remained for further analysis.
Base calling and demultiplexing was performed with the Illumina bcl2fastq 1.8.4 software
Peak calling was performed using MASC2 (v2.0.10)
bigwig files were generated from bedGraph files that were generated from MACS.
Genome_build: mm10
Supplementary_files_format_and_content: Peak bed files
Supplementary_files_format_and_content: bigwig files
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| Submission date |
Jan 23, 2018 |
| Last update date |
Jan 20, 2019 |
| Contact name |
Virginia Smith Shapiro |
| E-mail(s) |
shapiro.virginia1@mayo.edu
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| Phone |
507-293-0615
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| Organization name |
Mayo Clinic
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| Department |
Immunology
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| Street address |
200 First St SW
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| City |
Rochester |
| State/province |
MN |
| ZIP/Postal code |
55905 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE109529 |
HDAC3 restrains cytokine signaling during thymocyte development for the generation of CD4 T cells (ChIP-Seq) |
| GSE109531 |
HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment |
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| Relations |
| BioSample |
SAMN08386957 |
| SRA |
SRX3593545 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2945866_KO_Sel_input.bw |
301.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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