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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 31, 2021 |
| Title |
Ikaros_ChIP-seq |
| Sample type |
SRA |
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| Source name |
Primary thymocytes
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| Organism |
Mus musculus |
| Characteristics |
cell type: thymocytes
tissue: thymus
strain: C57BL/6
chip antibody: rabbit anti-Ikaros
chip antibody vendor: non-commercial, REF: Kleinmann, E., Geimer Le Lay, A.-S., Sellars, M., Kastner, P., & Chan, S. (2008). Molecular and cellular biology, 28(24), 7465–7475. doi:10.1128/MCB.00715-08, PMID:18852286
treatment: none
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| Treatment protocol |
Primary mouse thymocytes were cultured for 5 hr prior to crosslinking with 1% formaldehyde for 10 min at room temperature. Excess formaldehyde was scavenged by the addition of glycine to a final concentration of 125 mM. Crosslinked chromatin was prepared from crude nuclear extracts by micrococcal nuclease treatment (5 gel units/million cells), which was conducted for 15 min at 37°C in 10 mM Tris, pH 7.9, 10 mM NaCl, 4 mM CaCl2, 2% NP-40, and protease inhibitors, and stopped by the addition of 5 mM EDTA. After adding SDS to 0.1% and adjusting the NaCl to 150 mM, chromatin was further sheared by sonication, 15 cycles of 15 sec each (Branson 450 Digital Sonifier equipped with a stepped micotip and operated at 20% power), to an average fragment length of 180 bp. Genomic DNA was prepared from fragmented chromatin directly (input samples) or after immunoprecipitation (ChIP-seq samples), by reversing the chemical crosslinking at 65°C for 12 hr.
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| Growth protocol |
Primary mouse thymocytes were isolated from 5-8 week old mouse thymi and cultured in RPMI-1640 media supplemented with 5% fetal bovine serum and 100 units/ml penicillin-streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq and input libraries were prepared following Illumina's protocol with some modifications. The precipitated DNA was blunted, phosphorylated, and ligated to single-end adapter dimers, and then amplified by PCR [30 sec at 98°C; (10 sec at 98°C, 30 sec at 65°C, 30 sec at 72°C) x 14 cycles; 5 min at 72°C]. Excess PCR primers and adapter dimers were removed using AMPure beads (Agencourt Biosciences Corporation). DNA library size selection was performed by excising the 250-350 bp region from a 2% agarose gel followed by purification using a QIAquick Gel Extraction Kit (Qiagen).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Sample 4
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| Data processing |
base-calling: samples: BCL11B_ChIP-seq, BCL11B-PA_ChIP-seq, Input_BCL11B, Input-2; software: Illumina pipline; software version: 1.4.
base-calling: samples: Ikaros_ChIP-seq, Input-4; software: Illumina pipline; software version: 1.3.
base-calling: sample: Mi2beta_ChIP-seq; software: Illumina pipline; software version: 1.6.
alignment: as 36 base reads, BWA read quality trimming "-q 15"; software: BWA; software version: 0.6.2.
post alignment output quality filter: MAPQ ≥ 20; software: samtools; software version: 0.1.18.
peak calling: q-value ≤0.05; software: MACS; software version: 2.0.10.20130306.
Genome_build: mm10: BCL11B_ChIP-seq, Input_BCL11B
Genome_build: mm10-female: Ikaros_ChIP-seq, Input-4, Mi2beta_ChIP-seq, Input-2
Supplementary_files_format_and_content: bigWig reports normalized abundance density (reads per million)
Supplementary_files_format_and_content: bigBed reports peak calls
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| Submission date |
Jul 28, 2014 |
| Last update date |
Dec 31, 2021 |
| Contact name |
Walter K Vogel |
| E-mail(s) |
vogelw@onid.oregonstate.edu
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| Organization name |
Oregon State University
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| Department |
College of Pharmacy
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| Lab |
Mark Leid
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| Street address |
1601 SW Jefferson Street
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| City |
Corvallis |
| State/province |
OR |
| ZIP/Postal code |
97331 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE59826 |
Identification of genomic associations of transcription factors BCL11B, Ikaros, and Mi2beta in primary mouse thymocytes |
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| Relations |
| BioSample |
SAMN02943032 |
| SRA |
SRX663236 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1446970_Ikaros.bw |
56.5 Mb |
(ftp)(http) |
BW |
| GSM1446970_Ikaros_peaks.bigBED |
245.0 Kb |
(ftp)(http) |
BIGBED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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