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| Status |
Public on Aug 31, 2021 |
| Title |
Daxx F/F Pu.1 F/F 24wpi H3K9me3 1 |
| Sample type |
SRA |
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| Source name |
Hematopoietic Stem/Progenitor Cell
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Bone marrow
genotype: Daxx F/F Pu.1 F/F Cre +/-
chip antibody: H3K9me3
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Primary hematopoietic stem/progenitor cells were isolated form bone marrow and Lineage-negative/c-Kit-positive (HSPCs) were enriched by MACS. Nuclei were isolated, washed and frozen in wash buffer according to the “Bench top CUT&Tag V.3” protocol (https://ww.protocols.io/view/bench-top-cut-amp-tag-bcuhiwt6).
For the actual CUT&Tag experiment, we followed the CUT&Tag-direct protocol described by Henikoff et al.74 using the CUTANA pAG-Tn5 enzyme (Epicypher). Briefly, aliquots of 30,000-45,000 native nuclei per reaction were bound to activated Concanavalin A beads. After successive incubations with primary antibody (overnight at 4°C) and secondary antibody (0.5-1 h) in wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and 1x protease inhibitor), the beads were washed and resuspended in pAG-Tn5 (1:20 dilution) in 300-wash buffer (wash buffer containing 300 mM NaCl) for 1 h. Incubations were performed at room temperature, except when otherwise stated, in volumes of 25-50 ul in low-retention PCR tubes. Tagmentation was performed for 1 h in 300-wash buffer supplemented with 10 mM MgCl2. Following tagmentation, beads were washed in 50 ul TAPS buffer (10 mM TAPS pH8.5, 0.2 mM EDTA), resuspended in 5 ul SDS release buffer (0.1% SDS, 10 mM TAPS pH8.5) and incubated for 1 h at 58°C. SDS was neutralized with 15 ul of 0.67% Triton-X100, and 4 ul of dual indexed primers from the “IDT for Illumina Nextera DNA UD Indexes Set A” (Illumina) as well as 25 ul of NEBNext High-Fidelity 2x PCR Master mix (NEB) were added. Gap filling and 18 cycles of PCR were performed, followed by clean-up with 65 ul of SPRIselect beads (Beckman Coulter).
CUT&Tag
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
DaxxFFPu1FF_24wpi_H3K9me3_cov.bw
2485
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| Data processing |
Illumina Casava 1.8.4 for base calling and demultiplexing
Sequencing reads were adapter trimmed using Cutadapt 1.13 and aligned against mm10 (GENCODE release M14) using STAR aligner 2.5.3 with the following options: --alignIntronMax 1 --alignMatesGapMax 1800.
Reads mapping to the the mitochondrial genome and non-uniquely were removed. In addition, read duplicates were removed and start position of reads was offset by +4 bp if mapping to the positive strand and -5 bp if mapping to the negative strand.
For peak calling the ENCODE ATAC-seq analysis pipeline 0.3.4 was used.
Peak files were sorted by coordinate and overlapping peaks were merged with bedtools merge.
Identification of differentially accessible sites using diffreps with 600 bp window size.
Genome_build: mm10
Supplementary_files_format_and_content: Bed/NarrowPeak file with merged ATAC-seq peak positions and/or tab-delimited files with differentially accessible regions identified with diffreps
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| Submission date |
Mar 05, 2021 |
| Last update date |
Aug 31, 2021 |
| Contact name |
Paolo Salomoni |
| E-mail(s) |
paolo.salomoni@dzne.de
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| Organization name |
DZNE e.V.
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| Street address |
Sigmund-Freud-Str. 27
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| City |
Bonn |
| ZIP/Postal code |
53127 |
| Country |
Germany |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE119309 |
Aberrant chromatin landscape upon loss of the H3.3 chaperone Daxx leads to Pu.1-mediated neutrophilia and inflammation |
| GSE168367 |
Aberrant chromatin landscape upon loss of the H3.3 chaperone Daxx leads to Pu.1-mediated neutrophilia and inflammation [CUT&Tag] |
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| Relations |
| BioSample |
SAMN18168027 |
| SRA |
SRX10246522 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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