 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 19, 2022 |
| Title |
990_ATAC-seq |
| Sample type |
SRA |
| |
|
| Source name |
Primary AML
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Selected PBMC or BM cells
assay: ATAC-seq
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-seq was performed following the published protocol with minimal modification[Buenrostro et al., Curr Protoc Mol Biol, 2015]. Briefly, we also centrifuge down 50,000 cells, wash the cells with PBS, and perform nuclei extraction with cold lysis buffer. We added an extra step of washing the nuclei with another 500ul of lysis buffer to further remove mitochondrial DNA.
We then proceed with the transposition reaction (Illumina Tagment DNA Enzyme and Buffer Large Kit 20034198) and purification steps (QIAGEN MinElute PCR Purification Kit Cat No./ID: 28004) . We elute the DNA in 20ul elution buffer instead of 10ul elution buffer to increase recovery rate. Then we proceed with the PCR amplification step, where we use 20ul transposed DNA, 2.5ul of Nextera PCR primer 1 and 2.5ul of Nexteral primer 2, and 25ul KAPA HiFi HotStart Ready Mix master mix (KAPA KR0370). We use the PCR parameters indicated by the standard protocol with 11 cycles. We then perform size selection to remove small fragments using KAPA pure beads. We add 45ul KAPA beads to 50ul PCR solution, incubate for 15 minutes in room temperature, and use a magnet to capture the beads and discard the supernatant. We wash the beads with 200ul of 80% ethanol twice and remove the ethanol. We resuspend the beads in 20-50ul of prewarmed 10mM Tris-HCl, pH 8.0, incubate at 37 for 10 minutes, and use a magnet to recollect the supernatant as the final library. We use 1ul of the library to run on a 2% agarose gel to verify the footprint nucleosomes for successful assay. Libraries were sequenced as 150bp paired-end reads on platform Hiseq 4000 with 20 million raw read pairs per sample.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Reads were first trimmed by Cutadapt (version 2.4) with “-m 5 -e 0.2” options
Trimmed reads were then aligned to human genome reference GRCh38 with “bowtie2 -X2000 --mm” options
Mapped reads were deduplicated using Picard MarkDuplicates tool with “VALIDATION_STRINGENCY=LENIENT” option
Read alignments were shifted +4bp on “+” strand and -5bp on “-” strand to account for Tn5 insertion before peak calling.
Peaks were called by MACS2 with “--shift -75 --extsize 150 --nomodel -B --SPMR --keep-dup all --call-summits” options, and filtered against the ENCODE hg38 blacklist.
Genome_build: hg38
Supplementary_files_format_and_content: Peak, ATAC-seq peaks called by MACS2, filter by p value < 1e-5
|
| |
|
| Submission date |
Jun 09, 2020 |
| Last update date |
Jul 19, 2022 |
| Contact name |
Feng Yue |
| Organization name |
Northwestern University
|
| Department |
Biochemistry and Molecular Genetics
|
| Street address |
303 E Chicago Ave. Simpson Querrey 7-518
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE152134 |
Subtype-Specific and Structure Variation-Induced Chromatin Spatial Reorganization in Acute Myeloid Leukemia [ATAC-Seq] |
| GSE152136 |
Subtype-Specific and Structure Variation-Induced Chromatin Spatial Reorganization in Acute Myeloid Leukemia |
|
| Relations |
| BioSample |
SAMN15190111 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4604270_990_ATAC-seq.10bp.RPGC.bigwig |
157.5 Mb |
(ftp)(http) |
BIGWIG |
| GSM4604270_990_ATAC-seq.MACS2.filt.peaks.txt.gz |
1.0 Mb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
|
|
|
|
 |
