|
| Status |
Public on Apr 06, 2017 |
| Title |
Mac-24h-siPU Rep2 RNA-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 either a human non-targeting siRNA (Dharmacon Inc, D-001206-13-05) or 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 |
total RNA |
| Extraction protocol |
RNA quality was evaluated for all samples using an Agilent 2100 Bioanalyzer. Samples with a RIN > 9 were used to build RNA-seq libraries from 50,000 cells using the Smart-seq2 protocol (Picelli et al., 2013).
Reads were sequenced as 40 bp paired-end dual indexed reads using Illumina’s NextSeq 500
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
RNA-seq reads were mapped to the hg38 reference genome using STAR (Dobin et al., 2013) aligner.
Reads were mapped to Gencode version 20 gene annotations using Cufflinks (Trapnell et al., 2010).
FPKM and counts were normalized for batch effects using Combat (Johnson et al., 2007).
Genome_build: hg38
Supplementary_files_format_and_content: FPKM
|
| |
|
| Submission date |
Sep 18, 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 |
| GSE87055 |
Dynamic gene regulatory networks of human myeloid differentiation [RNA-seq_siRNA] |
|
| Relations |
| BioSample |
SAMN05786738 |
| SRA |
SRX2174265 |
