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Sample GSM6588378

Query DataSets for GSM6588378

Status

Public on May 23, 2023

Title

Kmt2a-MEP

Sample type

SRA

Source name

Bone-Marrow

Organism

Mus musculus

Characteristics

tissue: Bone-Marrow
strain: C57BL6
cell type: GMP
genotype: Wild-type
treatment: NA
chip antibody: Bethyl A300-086A

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

Description

MEP_Kmt2a_ChIP11_peaks.narrowPeak

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