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Sample GSM5893295

Query DataSets for GSM5893295

Status

Public on Nov 09, 2022

Title

tam4_ATAC-seq

Sample type

SRA

Source name

Splenocytes

Organism

Mus musculus

Characteristics

cell type: Lin- c-Kit+ GFP(AML cells) were sorted
treatment: GFP+Lin-c-Kit+Usp18+/D
genotype: Usp18+/f UBCER-Cre AE9a leukemia cells recipient mice were injected with tamoxifen

Treatment protocol

Usp18+/f UBCER-Cre AE9a leukemia cells were transplanted into recipient mice. After mice become sick, oil or tamoxifen were injected to heterozygously deplete Usp18. Three days after first injection, Lin- c-kit+ AML cells were sorted from mouse splenocytes.

Growth protocol

Lin- c-Kit+ GFP(AML cells) were sorted

Extracted molecule

genomic DNA

Extraction protocol

Permeabilized nuclei were obtained by resuspending cells in 250 µL Nuclear Permeabilization Buffer (10 mM Tris-HCL [pH 7.5], 10 mM NaCl, 3mM MgCl2, 0.1% Tween-20 [Sigma], 0.1% IGEPAL-CA630 [Sigma] and 0.01% Digitonin [Promega] in water) and incubating for 10 minutes on a rotator at 4°C. Nuclei were then pelleted by centrifugation for 5 minutes at 500 x g at 4°C. The pellet was resuspended in 20 µL ice-cold Tagmentation Buffer (33 mM Tris-acetate [pH 7.8] [BP-152, Thermo Fisher Scientific], 66 mM K-acetate [P5708, Sigma], 11 mM Mg-acetate [M2545, Sigma], 16% DMF [DX1730, EMD Millipore] in Molecular biology water [46000-CM, Corning]). An aliquot was then counted to determine nuclei concentration. Approximately 50,000 nuclei were resuspended in 10 µL ice-cold Tagmentation Buffer and incubated with 1 µL Tagmentation enzyme (FC-121-1030; Illumina) at 37°C for 30 minutes while shaking at 500 rpm. The tagmentated DNA was purified using the MinElute PCR purification kit (28004, Qiagen).
The libraries were amplified using NEBNext High-Fidelity 2X PCR Master Mix (M0541, NEB) with primer extension at 72°C for 5 minutes and denaturation at 98°C for 30 seconds, followed by 8 cycles of denaturation at 98°C for 10 seconds, annealing at 63°C for 30 seconds, and extension at 72°C for 60 seconds. Amplified libraries were then purified using the MinElute PCR purification kit (28004, Qiagen) and two size selection steps were performed using SPRIselect bead (B23317, Beckman Coulter) with 0.55X and 1.5X bead-to-sample volume ratios, respectively.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Data processing

Preprocessing: FASTQ files from sequencing experiments were mapped to the mm10 genome. Bowtie2 with default parameters was used to map ATAC-seq experiments (Langmead and Salzberg, 2012). HOMER was used to convert aligned reads into “tag directories” for further analysis (Heinz et al., 2010).
ATAC-seq experiments were performed in replicates. Peaks were called with HOMER for each data set using findPeaks with parameters -L 0 -C 0 -fdr 0.9 -minDist 200 - size 200. Peaks were combined using HOMER mergePeaks for downstream analysis. The pooled data from two replicates was used for track visualization using the UCSC Genome Browser.
Genome_build: mm10
Supplementary_files_format_and_content: Tab delimited text files of overall merger of all identified peaks.

Submission date

Feb 11, 2022

Last update date

Nov 09, 2022

Contact name

Kei-ichiro Arimoto

E-mail(s)

[email protected]

Organization name

UCSD Moores Cancer Center

Street address

3855 Health Sciences Dr