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Status |
Public on Mar 13, 2020 |
Title |
Mof-/+ HSC MOF IP |
Sample type |
SRA |
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|
Source name |
Bone Marrow
|
Organism |
Mus musculus |
Characteristics |
tissue: HSCs genotype: Mof -/+ strain: C57BL/6
|
Growth protocol |
Mouse bone marrow (BM) cells were isolated from pooled femora, tibiae, ilia and vertebrae by fluxing and crushing in cold PBS 2%FCS using a mortar and pistil. Lysis of erythrocytes was performed using ACK Lysing Buffer (Thermo Fisher Scientific). To deplete lineage-positive cells we used the MojoSort™ Mouse Hematopoietic Progenitor Cell Isolation Kit (Biolegend#48003). Total BM was incubated with biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads. Magnetically labeled fraction was retained using a magnetic separator and untouched hematopoietic stem/progenitor cells (HSPC) were collected by decanting the liquid in a clean tube. 15000 HSCs (LSK+CD34-Flt3-) and 30.000 MEPs (Linegae-Sca-1-cKit+CD34-IL7r-CD16/32-)were sorted by a FACS Fusion II (Becton Dickinson). HPC7 were cultured in Iscove Modified Medium supplemented with 10% FCS and 10 ng/mL of SCF.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. The library was completed and amplified through a PCR following the DeepSeq using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina default instructions (NEB #E7645). ChIP libraries were sequenced using an Illumina HiSeq 3000.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 3000 |
|
|
Data processing |
Genome alignment (mm10) by Bowtie2 version 2.3.0.1 (Langmead and Salzberg, 2012) using default parameters Quality control was assessed by deeptools Calling Peaks by MACS2 software (Feng et al., 2012), in which we settled bandwidth was settled for 300, lower mfold bound as 5, upper mfold bound as 500. Peak detection was based on q-value (minimum FDR cutoff as 0.05). For build model we selected the shifting mode. For normalization based on log 2 fold changes IP/Input deeptool Bamcompare (Galaxy Version 2.5.1.0.0) was used Coverage files were generate with bamCoverage module (Galaxy version 2.5.1.0.0) normalized coverage to 1x and effective geno size for mm10 Genome_build: mm10 Supplementary_files_format_and_content: Score represent the log2/fold change, wig we generated with bamCoverage module from deeptools software, Heatmap was generated eith the plotHeatmap module from deeptools and summary plots were generated with plotProflie module from deeptoools
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Submission date |
Nov 20, 2017 |
Last update date |
Mar 13, 2020 |
Contact name |
Asifa Akhtar |
E-mail(s) |
akhtarlab_data@ie-freiburg.mpg.de
|
Organization name |
Max Planck Institute of Immunobiology and Epigenetics
|
Department |
Chromatin Regulation
|
Lab |
Akhtar Lab
|
Street address |
Stuebeweg 51
|
City |
Freiburg |
ZIP/Postal code |
79108 |
Country |
Germany |
|
|
Platform ID |
GPL21493 |
Series (2) |
GSE107151 |
Genome-wide maps of chromatin state in HSC, HPC7 and lineage-committed cells |
GSE107154 |
Decoding the regulatory circuit by epigenetic regulator MOF in haematopoiesis |
|
Relations |
BioSample |
SAMN08045393 |
SRA |
SRX3409003 |