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Sample GSM1282506 Query DataSets for GSM1282506
Status Public on Sep 04, 2014
Title wildtype-input-2
Sample type SRA
 
Source name wildtype-input
Organism Mus musculus
Characteristics strain background: C57BL/6
genotype/variation: wild type
age: 4 weeks
tissue: thymus
cell type: mature mTEC
facs profile: CD45- EpCAM+ MHCIIhi Ly51- UEA+
Treatment protocol None.
Growth protocol C57BL/6 mice were obtained from Janvier (St Berthevin, France). Mice were maintained under specific pathogen-free conditions and according to Swiss federal and institutional regulations.
Extracted molecule genomic DNA
Extraction protocol Mature mTEC were isolated and sorted according to their cell surface phenotype. Fragmented thymi were digested repeatedly for 15-20 min at 37 °C with 1mg/ml Collagenase/Dispase (Roche Diagnostic) and 100µg/ml DNaseI (Roche Diagnostic) in HBSS containing 2% FCS (Perbio), to obtain single cell suspensions. After the final digest, cells were pooled and labelled with biotinylated anti-EpCAM for positive enrichment by AutoMACS system (Miltenyi Biotec), and stained using the following directly labelled antibodies and reagents: FITC-anti-IAb (clone AF6-120.1, BioLegend), PE-anti-Ly51 (clone 6C3, BioLegend), PECy7-anti-CD45 (clone 30-F11, BioLegend), APC-Cy7-anti-I-A/E (clone M5/114.15.2, BioLegend), biotinylated anti-EpCAM (clone G8.8, DSHB, University of Iowa), Streptavidin-labelled PerCP-Cy5.5 (BioLegend) and Cy5-UEA1 (Vector Laboratories). The cells were exposed to 4’, 6-diamidino-2-phenylindole (DAPI) to identify dead cells and then sorted by flow cytometry (FACSAira, BD Biosciences) achieving a TEC purity of over 93%. Chromatin immunoprecipitation (ChIP) was performed as previously described (Adli M, Bernstein BE. 2011. Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq. Nat Protoc 6(10): 1656-1668) with the following modifications: protein G magnetic beads (Dynabeads, Life Technologies) were used to capture antibody-chromatin complexes. The captured complexes were washed four times with wash buffer (0.1 % SDS, 1 % Triton X-100, 0.1 % deoxycholate, 20 mM Tris- HCl (pH 8), 1 mM EDTA, 0.5 mM EGTA) containing 150 mM NaCl, once with wash buffer containing 500 mM NaCl, and finally once with LiCl wash buffer (250 mM LiCl, 1 % NP-40, 1% deoxycholate, 10 mM Tris- HCl (pH 8), 1 mM EDTA). Antibodies used were anti-H3K4me3 (ab8580, Abcam) and anti-H3K27me3 (#9733, Cell Signalling).
ChIP-seq libraries were prepared with TruSeq ChIP Sample Preparation Kit (Illumina) and 50bp, paired-end reads were sequenced on an Illumina-HiSeq. For more information contact the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics, Oxford.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
 
Data processing (1) Alignment: 50bp, paired-end reads were mapped to the mouse genome (mm9) using bwa (version 0.5.9) with the options "aln -l 25 -k 2 -n 0.1 -q 20 "
(2) Duplicate read pairs were removed using Picard with the command "MarkDuplicates REMOVE_DUPLICATES=true"
(3) Normalised Bedgraph files were prepared for ChIP and input samples using the macs2 (https://github.com/taoliu/MACS/) algorithm with the "callpeak -B --SPMR" options.
(4) For each sample, enrichment over input was calculated using the macs2 algorithm with the "bdgcmp -m FE" options.
(5) After conversion to Wig format, the average enrichment over replicates was caclulated using the "wigmath.Average" function provided by java-genomics-toolkit
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files containing the (average) enrichment over input for the ChIP'ed histone modifications.
 
Submission date Dec 06, 2013
Last update date May 15, 2019
Contact name Stephen Sansom
E-mail(s) stephen.sansom@kennedy.ox.ac.uk
Organization name Kennedy Institute of Rheumatology
Department NDORMS
Lab Sansom
Street address Roosevelt Drive
City Oxford
State/province Oxfordshire
ZIP/Postal code OX3 7FY
Country United Kingdom
 
Platform ID GPL17021
Series (2)
GSE53109 Chromatin state in thymic epithelial cells [ChIP-seq]
GSE53111 Aire secures the transcription of polycomb marked genes to complete a comprehensive program of promiscuous gene expression in the thymic epithelial cell lineage
Relations
BioSample SAMN02438233
SRA SRX388797

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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