 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 31, 2020 |
| Title |
S001255_Tet2pp_n31_GFPneg_W31m_Input-11032 |
| Sample type |
SRA |
| |
|
| Source name |
Tet2+/+_input DNA
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J Tet2LacZ
genotype/variation: Tet2+/+
cell type: Primary murine mast cells derived from bone marrow
chip antibody: none (input)
|
| Treatment protocol |
Primary mast cells cultures were fixed by the addition of 1/10 volume of freshly made cross- linking solution (11% formaldehyde, 100mM NaCl, 1mM EDTA, 0.5 mM EGTA, 50mM Hepes pH 7.8) directly to cell medium and incubation at room temperature (RT) for 10 minutes. Formaldehyde was then quenched for 5 minutes at RT by the addition of 2.5M Glycine solution to a final concentration of 125mM. Fixed cells were washed twice with cold PBS, counted, and transferred in 50ml Falcon tubes. Cells were then pelleted by centrifugation at 700g for 5 minutes at 4°C, snap-frozen in liquid nitrogen and stored at −80°C for storage or shipment on dry-ice. Each batch of 10 million cells was lysed and processed using reagents and according to the protocol provided by the ChIP-IT High Sensitivity® (HS) Kit (Active Motif).
|
| Growth protocol |
Primary mast cells were derived from bone marrow of either Tet2+/+ or Tet2-/- C57BL/6J Tet2LacZ mice, isolated and cultured in OptiMEM/10% FCS (Life Technologies) media supplemented with 10 ng/ml IL3 (Peprotech), 50ug/ml Penicillin/streptomycin, and 1% beta-mercaptoethanol (both Life Technologies) on tissue culture treated plastic, at 37°C, 5% CO2. Non-adherent cells were selected for 15 days under these conditions, at which point cultures were infected with lenti-virus carrying the KitD816V allele and GFP (ires). For infection, we used a single 2h spinoculation in the presence of 6μg/ml polybrene. Infected cells were further expanded for 15 days, for a total of 30 days of in vitro culture, and then GFP-negative and GFP-positive fractions (cultures were 40-80% GFP-positive) were FACS sorted on FACSAria using DIVATM (Becton- Dickinson) software. Sorted cultures were expanded for 1-2 months in growth media. Fresh media was added to all cultures 1 day before fixation for ChIp. Fixed cell pellets were snap frozen in liquid nitrogen and stored at -80 degrees celcius.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The nuclei/chromatin suspension was sonicated using a Bioruptor Pico Sonnicator (Diagenode) for 20 cycles at 4°C. Sheared chromatin size was confirmed to be between 100-800 base-pairs on average by agarose gel. Two million cell equivalents were used for each ChIP. The following antibodies were used: H3K4me1 (Abcam, ab8895), H3K27ac (Abcam, ab4729), PU.1 (Santa Cruz Biotechnology, sc-352), Erg-1/2/3 (Santa Cruz Biotechnology, sc-354) and antibodies against total Stat5 (Santa Cruz, sc-835). ChIP samples were quantified using the Qubit Fluorometric quantification System (ThermoFisher) and using a DNA high sensitivity DNA kit on a Bioanalyzer (Agilent).
ChIP-seq libraries were made using the Multiplex Library Preparation Kit (Diagenode). Nine cycles of PCR were used for all libraries. After library DNA was purified, library concentration was measured by Qubit, and size distribution was determined by Bioanalyzer. Each ChIP-seq library was sequenced for single-end reads of Illumina HiSeq 4000 (between 100-800 bp read length). Biological replicates for ChIP-seq were performed on independently derived primary mast cell cultures from 2 different mice for each sample and both sequenced.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
S001255_Tet2pp_n31_GFPneg_W31m_Input-11032_R1_001
|
| Data processing |
Sequencing quality control was done with FastQC v0.11.6.
Cleaning up raw sequencing reads were performed with sickle v.0.6.3 (-q 20 , -l 25 parameters).
Cleaned Reads were mapped with the Bowtie2 V2.3.4.1 software to the mm10 (GRCm38), with on average 37 million, 35 million,38 million aligned reads/sample for PU.1, H3K27ac, H3K4me1 respectively.
SAM files where converted to BAM files and indexed using samtools V1.7.
The peak calling was carried out using MACS version 2.1.1.20160309 software (-q 0.01 for PU.1 ChiP, --broad for H3K27ac and H3K4me1 ChIP).
BedGraph control subtracted files were obtained using bdgcmp from same version of MACS.
Finaly BedGraph files were sorted and converted to bigwig files using bedGraphToBigWig V.4 for IGV visualization purpose.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files were generated using bedGraphToBigWig V.4 for IGV visualization purpose.
|
| |
|
| Submission date |
Nov 19, 2018 |
| Last update date |
Dec 31, 2020 |
| Contact name |
erinn soucie |
| E-mail(s) |
erinn.soucie@inserm.fr
|
| Organization name |
CRCM
|
| Street address |
27 Boulevard Lei Roure
|
| City |
MARSEILLE |
| ZIP/Postal code |
13009 |
| Country |
France |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE122686 |
Tet2 is required for Socs activation in immune response cells |
| GSE122700 |
Tet2 is required for Socs activation in immune response cells [ChIP-seq] |
|
| Relations |
| BioSample |
SAMN10441246 |
| SRA |
SRX5026261 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
|
|
|
|
 |
