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Sample GSM8101643

Query DataSets for GSM8101643

Status

Public on Apr 17, 2024

Title

Patient SU042 Diagnosis timepoint Blast replicate 1 ATACseq Sample

Sample type

SRA

Source name

AML

Organism

Homo sapiens

Characteristics

tissue: AML
individual: Patient SU042
timepoint: Diagnosis
cell type: Blast

Extracted molecule

genomic DNA

Extraction protocol

Sorted AML blasts were prepared for ATAC-seq as previously described54. 5,000-50,000 cells were washed in cold FACS buffer and spun at 4°C for 5 minutes at 500 rcf in a fix-angled centrifuge. Cell pellets were then resuspended in 50 uL of ATAC-seq resuspension buffer (RSB: 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water) with 0.1% NP40, 0.01% digitonin, and 0.1% Tween-20 and incubated on ice for three minutes. After lysis, 1 mL of ATAC-seq RSB with 0.1% Tween-20 was added and tubes were inverted six times to mix. Isolated nuclei were then spun at 4°C for 10 minutes at 500 rcf in a fix-angled centrifuge. Supernatant was removed and nuclei were resuspended in 50 uL transposition mix (25uL 2xTD buffer, 2.5 uL Tn5 transposase (100 nM final), 16.5 uL PBS, 0.5 uL 1% digitonin, 0.5 uL 10% Tween-20, and 5 uL nuclease-free water).
Transposition reactions were incubated at 37°C for 30 minutes in a thermomixer with shaking at 1000 rpm. Reactions were cleaned up using Qiagen MinElute Reaction Cleanup kits and processed as previously described. All libraries were amplified with a target concentration of 20 μl at 4 nM, which is equivalent to 80 femtomoles of product.
All libraries were sequenced on an Illumina NextSeq with 75 bp paired end reads.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

Patient SU042 Diagnosis timepoint Blast replicate 1 ATACseq Sample
bulk_count_matrix_raw.rds
bulk_gene_scores.rds

Data processing

ATAC-sea data was processed based on workflows similar to Corces et al. Science 201826. First, read trimming and quality control was performed using trim_galore using default parameters. Reads were aligned to the GRCh38 reference genome using bowtie2 and the --very-sensitive option. Aligned reads were converted to bam format and sorted using SAMtools, and deduplicated using Picard MarkDuplicates. Reads were subsequently indexed and mitochondrial reads were removed using SAMTools. TagAlign bed files from technical replicate libraries were generated using bedtools, shifted to find ATAC-seq cut sites, and pooled. Peaks were called using MACS2 using p-value cut-off of 0.01 and peaks falling within black list regions (https://sites.google.com/site/anshulkundaje/projects/blacklists) were removed. A consensus peak set was generated using methods from Corces et al. Science 201828. Briefly, called peaks were merged by merging all peak bed files and using an iterative removal process to resolve overlapping peaks. This approach resulted in a consensus set of 115,551 500bp fixed-width peaks covering accessible regions represented in at least 2 or more samples. Count matrices were generated using the consensus peak set, and the number of Tn5 cut sites in each peak were calculated using the CountOverlaps function from the GenomicRanges package in R.
Assembly: Processed data files aligned to hg38 reference genome.
Supplementary files format and content: R data file containing peak counts or gene accessibility scores for each sample.

Submission date

Feb 23, 2024

Last update date

Apr 17, 2024

Contact name

Ravindra Majeti

E-mail(s)

rmajeti@stanford.edu

Organization name

Stanford University

Lab

Ravindra Majeti