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| Status |
Public on Mar 29, 2013 |
| Title |
M_MAC_Donor2 |
| Sample type |
SRA |
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| Source name |
CD14+ monocytes
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| Organism |
Homo sapiens |
| Characteristics |
cell type: macrophage
treatments: M-CSF
donor: Donor2
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| Growth protocol |
CD14+ monocytes were isolated from healthy donor peripheral blood enriched leukopacks (Blood Bank, National Institute of Health, Bethesda, MD, USA) using MACS CD14 MicroBeads (Miltenyi Biotec, Auburn, CA, USA). Monocytes were differentiated into macrophages using 25 ng/ml M-CSF (R&D Systems, Minneapolis, MN, USA) (M-Mac) or a combination of M-CSF and 100 ng/ml IL-27 (R&D Systems) (I-Mac). Initially the monocytes were in Macrophage serum free media (Invitrogen, Carlsbad, CA, USA) which was replaced after three days with DMEM (Invitrogen) with 10% FBS (HyClone Laboratories, Logan, UT, USA), 25 mM HEPES (Quality Biology, Gaithersburg, MD, USA) and 5 µg/ml Gentamicin (Invitrogen) containing the same concentration of IL-27. Cells were incubated at 37°C in a 5% CO2 incubator for a total 7 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Qiagen miRNeasy kit (Qiagen, Hilden, Germany). Prior to RNA sequencing, samples from Donor 1 and 2 were subjected to analysis by the Agilent Bioanalyzer RNA Nano chip (Agilent Technologies, Santa Clara, CA, USA) to confirm RNA purity and quality (all four samples had a RNA integrity number of 10).
Four microRNA libraries were prepared using Illumina TruSeq Small RNA Sample Preparation protocol according to the manufacturer’s instructions. Briefly, the specifically modified RNA 3’ adaptor, which has a high affinity for Dicer cleavage products, and 5’ adaptor were aligned to the gel purified small RNA, followed by reverse transcription and library enrichment by PCR amplification. The size, purity, and concentration of the final cDNA library were validated using an Agilent Bioanalyzer 2100. Samples were sequenced on GAIIx with 36 cycles using Illumina TruSeq v3 chemistry.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Basecalls performed using RTA 1.8.70.0
The raw reads were trimmed against Illumina adapters, and rRNA, primers, mitochondria contaminations, and filtered by minimum length 15, maximum length 32 and phred-score 20 using fastq-mcf (code.google.com/p/ea-utils/wiki/FastqMcf). Then the reads were collapsed to tags by our own perl script. Tags containing 1 read were treated as machine error and removed. The clean tags were mapped to the hg19 build of the human genome (UCSC Genome Browser) and human mature miRNA (miRbase v19) using novoalign (http://www.novocraft.com/) with 2 mismatches.
Qualified tags were inputted into the publicly available miRNA discovery program, miRDeep to identify novel miRNAs
Reads counts were calculated using our own scripts from novoalign mapping result, and then were normalized by DESeq
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include normalized value for each Sample ...
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| Submission date |
Mar 07, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Xiaojun Hu |
| Organization name |
SAIC_Frederick, INc
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| Department |
CSP
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| Lab |
LIB
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| Street address |
1050 Boyles St.
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| City |
Frederick |
| State/province |
MD |
| ZIP/Postal code |
21702 |
| Country |
USA |
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| Platform ID |
GPL10999 |
| Series (2) |
| GSE44953 |
Interleukin-27 treated human macrophages induce the expression of novel miRNAs which may mediate anti-viral properties (RNA-seq) |
| GSE44957 |
Interleukin-27 treated human macrophages induce the expression of novel miRNAs which may mediate anti-viral properties |
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| Relations |
| SRA |
SRX248548 |
| BioSample |
SAMN01974915 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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