NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM5137393 Query DataSets for GSM5137393
Status Public on Aug 31, 2021
Title Daxx F/F Pu.1 F/F 8wpi H3K9me3 2
Sample type SRA
 
Source name Hematopoietic Stem/Progenitor Cell
Organism Mus musculus
Characteristics strain: C57BL/6
tissue: Bone marrow
genotype: Daxx F/F Pu.1 F/F Cre +/-
chip antibody: H3K9me3
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
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
Description DaxxFFPu1FF_8wpi_H3K9me3_cov.bw
H3K9me3_DaxxFFPu1FFvsDaxxWtWt_8wpi_diffsite.txt
3394
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
 
Submission date Mar 05, 2021
Last update date Aug 31, 2021
Contact name Paolo Salomoni
E-mail(s) paolo.salomoni@dzne.de
Organization name DZNE e.V.
Street address Sigmund-Freud-Str. 27
City Bonn
ZIP/Postal code 53127
Country Germany
 
Platform ID GPL24247
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]
Relations
BioSample SAMN18167841
SRA SRX10246496

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

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap