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| Status |
Public on Jan 23, 2018 |
| Title |
L-DKO DP E2A |
| Sample type |
SRA |
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| Source name |
sorted cells from thymus
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| Organism |
Mus musculus |
| Characteristics |
strain: L-DKO mice (deficient in Id2 and Id3)
age: 3-5 weeks
tissue: thymus
cell type: double-positive (DP) thymocyte
sorting strategy: CD1dTet- CD4+ CD8+
chip antibody: anti-E2A antibody (V-18, Santa Cruz Biotechnology, Lot G0814)
molecule subtype: genomic DNA + crosslinked, bound protein
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Thymii were harvested from mice, and thymocytes were stained with appropriate antibodies for sorting. Following sort, Cells were fixed for 15 min in PBS containing 1.5 mM EGS(ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester)), followed by incubation for 15 min with 1% formaldehyde. Formaldehyde was quenched by incubation for 10 min with 0.2 M glycine, and then washed twice with PBS and pellets were frozen in liquid nitrogen. Crosslinked cells were lysed, washed and sonicated for 22 cycles using a Biorupter Plus sonicator. Lysates were cleared with dilution buffer, and incubated overnight at 4°C with magnetic beads bound with 15 μg of anti-E2A antibody (V-18, Santa Cruz Biotechnology, Lot G0814) or 3 μl anti-H3K4me2 antibody (Millipore, 07-030, Lot 2430486). Beads were washed multiple times with appropriate buffers. Bound complexes were then eluted from beads by incubation for 30 min at 65 °C with shaking in elution buffer. Crosslinks were reversed by incubation overnight at 65°C. RNA and proteins were digested, followed by DNA purification using a ChIP DNA Clean and Concentrator kit (Zymoresearch).
Libraries were prepared with the NEBNext primer set which involved applying ChIP DNA to end repair, A-tailing, adapter ligation, PCR amplification. They were then cleaned and size selected by AMPure beads (Agencourt)
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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 |
Reads were aligned to the mm9 genome using Bowtie software (version 1.1.2, parameters: --chunkmbs 128 --mm -m1 --best --strata -p4 -S -q)
Peaks were called using MACS (version 1.4.2, default parameters)
Peaks called by MACS were annotated by the NGS: Peak Annotation tool on Nebula (https://nebula.curie.fr/)
Genome_build: mm9
Supplementary_files_format_and_content: Bed and wig files were generated by MACS for visualization using the IGV tool
Supplementary_files_format_and_content: Scores in bed files represent peak scores assigned by MACS
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| Submission date |
Nov 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Yuan Zhuang |
| E-mail(s) |
yuan.zhuang@duke.edu
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| Phone |
919-613-7824
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| Organization name |
Duke University
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| Department |
Immunology
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| Lab |
Zhuang lab
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| Street address |
207 Research Drive
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27705 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE89847 |
Id proteins suppress E2A-driven iNKT cell development prior to TCR selection [ChIP-seq] |
| GSE89849 |
Id proteins suppress E2A-driven iNKT cell development prior to TCR selection |
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| Relations |
| BioSample |
SAMN06017337 |
| SRA |
SRX2348473 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2391480_LDKO_DP_E2AvsInput_AnnotatedPeaks_new.xlsx |
1.6 Mb |
(ftp)(http) |
XLSX |
| GSM2391480_LDKO_DP_E2AvsInput_peaks.bed.gz |
254.2 Kb |
(ftp)(http) |
BED |
| GSM2391480_LDKO_DP_E2AvsInput_treat.wig.gz |
223.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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