|
| Status |
Public on Apr 06, 2017 |
| Title |
Mac-24h-siPU Rep1 ATAC-seq |
| Sample type |
SRA |
| |
|
| Source name |
Macrophage
|
| Organism |
Homo sapiens |
| Characteristics |
induction: PMA
time: 24 hr
treatment: siRNA PU.1
|
| Treatment protocol |
Differentiation of HL-60 cells into macrophage (Murao et al., 1983), monocytes (Mangelsdorf et al., 1984) and neutrophils (Breitman et al., 1980) was performed as previously described. Additionally, monocytes (120 hours post-differentiation) were stimulated with PMA to induce differentiation into monocyte-derived macrophages. Briefly, 1x10e6 undifferentiated HL-60 cells were washed in 1X PBS and transfected with the Lonza nucleofector cocktail, 1μg of GFP plasmid and human PU.1 siRNA (Dharmacon Inc, E-010537-00-0005). HL-60 and differentiated myeloid cells were harvested 24 hours post-nucleofection. Cell viability was determined using trypan blue staining and counted using a hemocytometer prior to RNA isolation.
|
| Growth protocol |
HL-60 cells (ATCC) were grown in Modified Dulbecco's Medium in a final concentration of 20% FBS with penicillin antibiotics (1%). Cells were routinely cultured at a density of 1x10^6 cells/ml.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 50,000 HL-60 and differentiated cells were collected for ATAC-seq. ATAC-seq was performed as previously described (Buenrostro et al., 2013) with one amendment. DNA size exclusion was utilized after library generation to enrich for accessible chromatin ranging from 100-400bp.
ATAC-seq libraries as paired-end 43bp reads on the Nextseq 500 Illumina platform.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Library strategy: ATAC-seq
ATAC-seq reads were mapped to the hg38 reference genome using bowtie (Langmead et al., 2009). Using parameters (bowtie -S --trim3 7 --chunkmbs 3000 -p 32 -m 3 -v 2 --best --strata -X 3000)
BAM was generated from SAM file using samtools/0.1.18
BedTools genomeCoverageBed was used to convert BAM to bedGraph with hg38 chromosome sizes.
bedGraphToBigWig from UCSC was used to generate bigWig files.
Genome_build: hg38
Supplementary_files_format_and_content: bigwig
|
| |
|
| Submission date |
Sep 20, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ricardo Noel Ramirez |
| E-mail(s) |
ricardnr@uci.edu
|
| Organization name |
University of California Irvine
|
| Department |
Developmental and Cell Biology
|
| Street address |
2300 Biological Sciences III
|
| City |
Irvine |
| State/province |
California |
| ZIP/Postal code |
92697 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE79046 |
Dynamic gene regulatory networks of human myeloid differentiation |
| GSE87114 |
Dynamic gene regulatory networks of human myeloid differentiation [ATAC-seq_siRNA] |
|
| Relations |
| BioSample |
SAMN05791198 |
| SRA |
SRX2178697 |
