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Sample GSM2391482 Query DataSets for GSM2391482
Status Public on Jan 23, 2018
Title L-DKO NKT E2A
Sample type SRA
 
Source name sorted cells from thymus
Organism Mus musculus
Characteristics strain: L-DKO mice (deficient in Id2 and Id3)
age: 3-5 weeks
tissue: thymus
cell type: Invariant Natural Killer T (iNKT) cells
sorting strategy: CD1dTet+ TCRβ+
chip antibody: anti-E2A antibody (V-18, Santa Cruz Biotechnology, Lot G0814)
molecule subtype: genomic DNA + crosslinked, bound protein
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)
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 4000
 
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
 
Submission date Nov 15, 2016
Last update date May 15, 2019
Contact name Yuan Zhuang
E-mail(s) yuan.zhuang@duke.edu
Phone 919-613-7824
Organization name Duke University
Department Immunology
Lab Zhuang lab
Street address 207 Research Drive
City Durham
State/province NC
ZIP/Postal code 27705
Country USA
 
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
Relations
BioSample SAMN06017335
SRA SRX2348475

Supplementary file Size Download File type/resource
GSM2391482_LDKO_NKT_E2AvsInput_AnnotatedPeaks_new.xlsx 170.3 Kb (ftp)(http) XLSX
GSM2391482_LDKO_NKT_E2AvsInput_peaks.bed.gz 23.0 Kb (ftp)(http) BED
GSM2391482_LDKO_NKT_E2AvsInput_treat.wig.gz 348.7 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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