![](/coreweb/template1/pix/main_left_bg.gif) |
![](/coreweb/template1/pix/pixel.gif) |
GEO help: Mouse over screen elements for information. |
|
Status |
Public on May 23, 2023 |
Title |
IgG-DM |
Sample type |
SRA |
|
|
Source name |
Bone-Marrow
|
Organism |
Mus musculus |
Characteristics |
tissue: Bone-Marrow strain: C57BL6 cell type: Leukaemia cell genotype: Npm1c/Flt3-ITD treatment: Expanded with SCF, IL3, IL6 chip antibody: abcam ab171870
|
Treatment protocol |
Cells were dual-crosslinked with 3 mM DSG,DMA,EGS for 20 minutes at room temperature followed by 1% Formaldehyde for 5 more minutes. The reaction was quenched by adding Glycine to 125 mM final concentration, then cells were pelleted, washed twice with cold 0.5% BSA/PBS containing 1X Protease Inhibitors and frozen at -80C.
|
Growth protocol |
GMPs, Monocytes, MEPs, Erythroblasts and Bcells were freshly sorted from 12-14 week old C57BL6 mice bone marrow in 1X PBS + 0.01% BSA. Ex vivo Monocytes were expanded for 7 days with 20% FBS, 10 ng/ml GM-CSF, SCF, 5 ng/mL G-CSF, IL3, IL6, IL5, Flt3L, 2 ng/mL Tpo and 2 U/mL Epo. Leukaemia cells were expanded for 5 days with 5% FBS, 50 ng/ mL SCF, 10 ng/mL IL3 and IL6
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were lysed in hypotonic buffer (10 mM NaCl, 0,1% IGEPAL + Prot Inhibitors) and Chromatin was extracted by Sonication (Bioruptor Pico) in 0.5% SDS, 5mM EDTA + Prot Inhibitors. Then, chromatin fragments were extracted using 4 volumes of 25 mM HEPES, 185 mM NaCl, 1.25% Tx-100 + Protease Inhibitors. Next, samples were incubated for 10-12 hours with the relevant antibodies, then captured using 25 uL Magna ChIP Prot A+G and incubating for 2h at 4 ºC. Next, ChIPed DNA was released and reversed crosslinking by 45 min incubation with 2 uL of Proteinase K in ChIP Elution buffer followed by 1-hour incubation at 68 ºC. Finally, the ChIPed DNA was purified with a 2.2X SPRI Cleanup and quantified by Qbit. ChIP-seq libraries were prepared from 1-10 ng of DNA with NEB Next Ultra II kit, QCed for concentration (Qbit) and size (Tape-Station) and sequenced to 100 million reads per sample.
|
|
|
Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
NextSeq 1000 |
|
|
Data processing |
We followed the ChIP-seq nf-core pipeline ChIP-seq reads were trimmed with default parameters using Trim Galore v0.6.6 with Cutadapt v3.4 ATAC-seq reads were aligned to the GRCm38/mm10 reference genome assembly using Bowtie version 2.3.4.2 with parameters -X 1000 --no-discordant --no-mixed --very-sensitive We removed duplicated regions with Picard v2.25.4 and filtered out ENCODE blacklist regions v2.0 and non-interesting chromosomes We pooled replicates We pooled replicates and converted the paired BAM files to single-read bed format using function bamToBed from BEDTools v2.27.1 Peaks were called uing MACS v2.2.7.1 with parameters -f BAMPE--keep-dup all Assembly: GRCm38/mm10 Supplementary files format and content: bamfile, narrowPeak
|
|
|
Submission date |
Sep 16, 2022 |
Last update date |
May 23, 2023 |
Contact name |
Brian Huntly |
E-mail(s) |
bjph2@cam.ac.uk
|
Organization name |
University of Cambridge
|
Department |
Department of Haematology
|
Street address |
Hills Road
|
City |
Cambridge |
ZIP/Postal code |
CB2 0AW |
Country |
United Kingdom |
|
|
Platform ID |
GPL32159 |
Series (2) |
GSE213507 |
Comparative binding patterns of Chromatin Regulators in normal versus leukemic hematopoiesis [ChIP-Seq] |
GSE213513 |
Leukemic hematopoiesis |
|
Relations |
BioSample |
SAMN30889422 |
SRA |
SRX17605415 |
Supplementary data files not provided |
SRA Run Selector![Help](/coreweb/images/long_help4.gif) |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
![](/coreweb/template1/pix/main_right_bg.gif) |