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| Status |
Public on Dec 31, 2020 |
| Title |
Tet2KO control-Rep1 |
| Sample type |
SRA |
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| Source name |
Primary murine mast cells derived from bone marrow, Tet2-/- , Rep1
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J Tet2LacZ
cell type: Primary murine mast cells derived from bone marrow
genotype: Tet2-/-
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| Treatment protocol |
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 positive fractions (cultures were 40-80% GFP-positive) were FACS sorted on FACSAria using DIVATM (Becton- Dickinson) software for direct lysis and gDNA isolation.
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| Growth protocol |
Primary mast cells were derived from bone marrow of either Tet2+/+ or Tet2-/- C57BL/6J Tet2LacZ
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sorted GFP-positive/KitD816V and GFP-negative/control cell fractions were directly lysed and genomic DNA was isolated using the DNAeasy kit (Qiagen) for RRBS.
RRBS libraries were prepared as previously described (Auclair G. et al, Genome Biol. 15, 545, 2014). Briefly, we digested 100 ng of genomic DNA 5 hours with MspI (Thermo Scientific), followed by end-repair and A-tailing (Klenow fragment, Thermo Scientific) and ligation to methylated adapters (T4 DNA ligase, Thermo Scientific) in Tango 1X buffer. Fragments between 150 and 400 bp were excised from a 3% agarose 0.5X TBE gel with the MinElute kit (Qiagen) and bisulfite-converted with the EpiTect Bisulfite kit (Qiagen) using two consecutive rounds of conversion. Final RRBS libraries were amplified with 14 cycles of PCR.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Sequenced reads were trimmed using Trim Galore v0.4.2 (parameters --rrbs --paired -r1 30 -r2 30 -q 20 --length 20 --retain_unpaired --stringency 5).
Reads were aligned to the mouse mm10 genome using BSMAP v2.74 (parameters -v 2 -w 100 -r 1 -x 400 -m 30 -n 1 -D C-CGG).
We extracted methylation percent values as the ratio of the number of Cs over the total number of Cs and Ts with methratio.py from BSMAP v2.74 (parameters -z -u -g).
We then filtered CGs to have a minimum sequencing depth of 8x using custom developped scripts in R v3.0.0.
Genome_build: mm10
Supplementary_files_format_and_content: IGV files containing methylation scores for all CGs with at least 8x sequencing depth.
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| Submission date |
Nov 21, 2018 |
| Last update date |
Jan 01, 2021 |
| Contact name |
erinn soucie |
| E-mail(s) |
erinn.soucie@inserm.fr
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| Organization name |
CRCM
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| Street address |
27 Boulevard Lei Roure
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| City |
MARSEILLE |
| ZIP/Postal code |
13009 |
| Country |
France |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE122686 |
Tet2 is required for Socs activation in immune response cells |
| GSE122797 |
Tet2 is required for Socs activation in immune response cells [RRBS] |
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| Relations |
| BioSample |
SAMN10458804 |
| SRA |
SRX5043691 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3485532_Tet2KO_control-Rep1.igv.gz |
8.4 Mb |
(ftp)(http) |
IGV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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