 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 09, 2015 |
| Title |
Rough.4.1 |
| Sample type |
SRA |
| |
|
| Source name |
THP-1-derived macrophage
|
| Organism |
Homo sapiens |
| Characteristics |
bacterial infection: M. abscessus Rough (MAB-R)-infected
time point (hours post-infection): 4
|
| Treatment protocol |
Before the infection, the medium was replaced with fresh RPMI/10% FBS/2mM L-Glutamine medium without antibiotics and cells were infected with mycobacteria to achieve a multiplicity of infection (MOI) of 10:1 (bacteria : cells). Non infected control macrophages received culture media only. Both not-infected and infected macrophages were prepared in triplicate in 21 cm2 tissue culture dishes and incubated at 37°C. After 1hpi, the infected macrophages were washed with sterile PBS to remove the extracellular mycobacteria and reincubated for 4hpi or 24hpi in culture media supplemented with 60ug/mL of amikacin (Sigma) at 37°C 5% CO2 until the macrophages were harvested. This was necessary to inhibit the growth of the remaining extracellular bacteria. In preliminary experiments, we confirmed that, at this concentration, amikacin did not influence intracellular viability of both strains of MAB (data not shown).
|
| Growth protocol |
THP-1 cells were obtained from the American Type Culture Collection (ATCC, TIB-202). The cells were grown in suspension in 75 cm2 flasks in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life technologies), 1% penicillin/streptomycin (Life technologies) and 2 mM L-Glutamine (Sigma) at 37 °C in humidified CO2 incubator. Antibiotics were used in the growth medium of THP-1 cells to avoid contamination of the propagating cells. To differentiate THP-1 cells into macrophages, the cells were seeded in 21 cm2 tissue culture dishes (CellStar) at a density of 107 cells/dish and incubated with 25 ng/mL of phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 1, 4 and 24 hours post-infection, infected macrophages were washed twice with PBS and homogenized by adding Trizol reagent (Sigma). All the aliquots were stored at -80°C until required for RNA extraction. RNA extraction was performed by adding 200uL of chloroform (Sigma) to each sample and mixing by vortexing for 60 sec. The solution was then centrifuged for 15 min at 12000xg at 4°C. The aqueous phase was removed, transferred to a separate tube containing 500uL of isopropanol (Sigma), gently mixed and centrifuged 10 min at 12000xg at 4°C. The supernatant was discarded, the RNA pellet was resuspended in 1mL of 75% Ethanol, mixed by vortexing and centrifuged 5 min at 7500xg at 4°C. After discarding all the supernatants, the RNA was transferred to a miRNeasy mini kit column (Qiagen, Ldt.) and further purification steps were conducted according to the manufacturer’s instructions. In addition, the RNA was subjected to an on-column DNase treatment step (Qiagen Ltd.) to remove any DNA residues that could affect the downstream reaction. RNA quantity and quality was assessed using a NanoDrop TM 1000 spectrophotometer (Thermo Fisher Scientific Inc.) and on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip kit (Agilent Technologies, Ltd.). All samples displayed a 260/280 ratio greater than 2.0 and RNA integrity numbers (RIN) greater than 7,5.
Small RNA-seq libraries were prepared using the Illumina TruSeq Small RNA Sample Preparation Kit according to the manufacturer’s protocol using 1ug of total RNA (extracted from not-infected and infected samples not bead-beaten). Briefly, RNA adapters were ligated to the 5’ and 3’ ends of the small RNA molecules, followed by reverse transcription and 11 cycles of PCR amplification by using indexed primers. The libraries were size-selected on a 6% polyacrylamide gel for the desired size of 147-157 bp and then purified from the gel. The molarity and size of finished miRNA-seq libraries were quantified on an Agilent 2100 Bioanalyzer using an Agilent High Sensitivity DNA chip (Agilent technologies). Libraires were loaded at a concentration of 10pM onto the flowcell and were single-end sequenced on Illumina HiSeq 2000 or Illumina MiSeq.
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Basecalls were performed using Illumina CASAVA v1.8.2
Adapters were trimmed from the 3' ends of sequenced reads using Fastx-toolkit
Reads were aligned to the human reference genome using bowtie v1.1.2 using the mapper.pl script from miRdeep2, with the -d -n -e -h -j and -m flags
Read counts for miRNAs were obtained using the miRDeep2.pl script from miRdeep2, with mature and precursor human miRNA sequences from miRBase
Genome_build: GRCh38
Supplementary_files_format_and_content: Raw counts of the numbers of reads mapped to all miRNAs from miRBase
|
| |
|
| Submission date |
Sep 08, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Brendan J. Loftus |
| E-mail(s) |
brendan.loftus@ucd.ie
|
| Organization name |
University College Dublin
|
| Department |
School of Medicine & Medical Science
|
| Street address |
Conway Institute, University College Dublin
|
| City |
Dublin |
| ZIP/Postal code |
Dublin 4 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE72769 |
High-throughput small RNA-sequencing of human macrophages infected with Mycobacterium abscessus Smooth and Rough variants |
| GSE72822 |
High-throughput sequencing of human macrophages infected with Mycobacterium abscessus Smooth and Rough variants |
|
| Relations |
| BioSample |
SAMN04038665 |
| SRA |
SRX1203796 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
|
|
|
|
 |
