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| Status |
Public on Mar 26, 2013 |
| Title |
716 control 24h |
| Sample type |
SRA |
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| Source name |
Monocyte-derived macrophages
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| Organism |
Bos taurus |
| Characteristics |
strain: Holstein-Friesian
Sex: Female
age: Four years old
animal id: 716
cell type: Monocyte-derived macrophages
infection status: Control uninfected
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| Treatment protocol |
Culturing of M. bovis was performed in a Biosafety Containment Level 3 (CL3) laboratory and conformed to the national guidelines on the use of Hazard Group 3 infectious organisms. A clinical isolate of M. bovis, obtained from an infected Irish animal was supplied by Dr. Eamon Costello (Department of Agriculture, Fisheries and Food, Backweston, County Kildare) was used in the current study. M. bovis stocks for MDM-challenge experiments were cultured in Middlebrook 7H9 medium (DifcoTM, Becton-Dickinson, Dublin, Ireland) containing 10% (volume/volume) Middlebrook albumin-dextrose-catalase (ADC) enrichment (DifcoTM, Becton-Dickinson, Dublin, Ireland), 0.05% Tween 80 (Sigma-Aldrich, Dublin, Ireland) and 0.40% (weight/volume) sodium pyruvate (Sigma-Aldrich, Dublin, Ireland) at 37°C. For colony forming unit (cfu) counting Middlebrook 7H11 medium (DifcoTM, Becton-Dickinson, Dublin, Ireland) containing 0.40% (volume per volume) sodium pyruvate and 10% (volume/volume) Middlebrook (ADC) enrichment. Bacterial cultures were grown to mid-logarithmic phase as determined by spectrophotometric analysis prior to the challenge experiments using the purified bovine MDM. All challenge experiments including the preparation of non-challenge control samples were performed in the CL3 laboratory. MDM samples (seeded at 2 × 105 cells per well) were challenged with M. bovis (4 × 105 cells/well; based on bacterial cell counts performed using a Petroff Hausser chamber (Fisher-Scientific, Dublin, Ireland) [multiplicity of infection 2:1] and incubated at 37°C, 5% CO2 for 24 hours. For the non-challenged control samples at 24 hours, antibiotic-free culture media (RPMI 1640 medium containing 15% heat inactivated FCS and 1% NEAA only) was added to each well. After 2 hours post-challenge, the media from all 24 hours challenge experiments was replaced with 0.5 ml fresh antibiotic-free culture media per well and re-incubated at 37°C, 5% CO2 until MDM were required for harvesting. Challenged and non-challenged control MDMs were lysed and harvested using RLT/1% β-mercaptoethanol buffer (Qiagen Ltd., Crawley, UK) at the designated time points. For each treatment, MDM lysates from two culture plate wells were pooled and stored at ‑80°C until required for RNA extraction.
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| Growth protocol |
Seven age-matched (three-year old) Holstein-Friesian females were used. All animals were maintained under uniform housing conditions and nutritional regimens at the UCD Lyons Research Farm (Newcastle, County Kildare, Ireland). The animals were selected from an experimental herd without a recent history of bovine tuberculosis infection. This was confirmed using the single intradermal tuberculin test (SICTT) and the BoviGAM® IFN-g enzyme linked immunosorbent assay (ELISA; Prionics AG, Schlieren-Zurich, Switzerland) with all animals testing negative. These cattle also tested negative for Brucella abortus, M. avium subsp. paratuberculosis, Salmonella typhimurium, bovine herpesvirus 1 and bovine viral diarrhoea (BVD) virus. For monocyte isolation, 300 ml of whole blood was collected in acid citrate dextrose buffer (Sigma-Aldrich Ireland Ltd., Dublin, Ireland) in sterile bottles. Blood was layered onto Accuspin™ tubes containing Histopaque® 1077 (Sigma-Aldrich Ireland Ltd., Dublin, Ireland) and following density gradient centrifugation, peripheral blood mononuclear cells (PBMC) were collected. Contaminating red blood cells (RBC) were removed following resuspension and subsequent incubation of the PBMC in RBC lysis buffer (10mM KHCO3, 150mM NH4Cl, 0.1mM EDTA pH 8.0) for 5 minutes at room temperature. After incubation, PBMC were washed twice with phosphate-buffered saline (PBS) before resuspending cells in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA, Sigma-Aldrich Ireland Ltd., Dublin, Ireland). Monocytes were then isolated using the MACS® protocol and a human anti-CD14 antibody (Miltenyi Biotec Ltd., Surrey, UK), which has been shown to be cross-reactive with bovine monocytes. All procedures were carried out according to the manufacturers’ instructions. The identity and purity of monocytes was confirmed by flow cytometry using an anti-CD14 FITC labelled antibody. Purified monocytes were seeded at 1 × 106/ml in 24-well tissue culture plates in RPMI 1640 medium (Invitrogen Ltd., Paisley, UK) containing 15% heat inactivated foetal calf serum (FCS; Sigma-Aldrich Ireland Ltd., Dublin, Ireland), 1% non-essential amino acids (NEAA; Sigma-Aldrich Ireland Ltd., Dublin, Ireland), gentamicin (5 µg/ml; Sigma-Aldrich Ireland Ltd., Dublin, Ireland) and incubated at 37°C, 5% CO2. Following 24 hours incubation (day one) the media was replaced with 1ml fresh antibiotic-containing media to remove any non-adhered cells. On day three, media was replaced with 1 ml antibiotic-free culture media (RPMI 1640 medium containing 15% heat inactivated FCS and 1% NEAA only). To ensure that the same number of MDM were subjected to different treatments between experiments, cells were dissociated on day five using a non-enzymatic cell dissociation solution (Sigma-Aldrich Ireland Ltd., Dublin, Ireland), counted and then re-seeded at 2 × 105 cells/well in 24-well tissue culture plates (Sarstedt, County Wexford, Ireland) using antibiotic-free culture media. By day eight, 80-100% confluent monolayers of MDM were generated that displayed the characteristic macrophage morphology as confirmed by Giemsa staining (data not shown). Day eight MDM were used for the in vitro challenge experiments with M. bovis.
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| Extracted molecule |
total RNA |
| Extraction protocol |
All RNA extractions were performed in the CL3 laboratory using an RNeasy kit incorporating an on-column DNase treatment step (Qiagen Ltd., Crawley, UK) according to the manufacturer’s instructions. RNA quantity and quality was assessed using both the NanoDropTM 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip kit (Agilent Technologies, Cork, Ireland). All samples displayed a 260/280 ratio greater than 2.0 and RNA integrity numbers (RIN) greater than 8.5.
In total, 14 strand-specific RNA libraries for high-throughput sequencing were prepared (seven libraries for each treatment: M. bovis-infected and control samples) using 200 ng of total RNA. Total RNA was first heated at 65°C for 5 min to disrupt any secondary structure. Purification of poly(A) RNA was performed using a Dynabeads® mRNA DIRECTTM Micro Kit according to the manufacturer’s instructions (Invitrogen™/Life Technologies Ltd., Paisley, UK). Purified poly(A) RNA was then fragmented using 1× RNA Fragmentation Reagent (Ambion®/Life Technologies Corporation, Warrington, UK) for 5 min at 70°C and precipitated using 68 mM sodium acetate pH 5.2 (Ambion), 227 ng/μl glycogen (Ambion) and 30 μl of 100% ethanol (Sigma-Aldrich Ltd., Dublin, Ireland). Pellets were washed with 80% ethanol, air-dried for 10 min at room temperature and re-suspended in 10.5 µl DNase- and RNase-free water. Synthesis of first strand cDNA was performed by incubating fragmented RNA with 261 mM Random Hexamer Primers (Invitrogen), 1× first strand buffer (Invitrogen); 10 mM DTT (Invitrogen); 0.5 mM dNTPs; 20 U RNaseOUTTM Recombinant Ribonuclease Inhibitor; and 200 U SuperScript® II Reverse Transcriptase (Invitrogen) at 25°C for 10 min, at 42°C for 50 min, and 70°C for 15 min. First strand synthesis reaction mixtures were purified using MicroSpinTM G-50 columns according to the manufacturer’s instructions (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, UK). Second strand cDNA synthesis, involving the incorporation of uracil, was performed by adding the first strand cDNA synthesis reaction to a second strand reaction mix consisting of 0.065× first strand buffer (Invitrogen); 1× second strand buffer (Invitrogen); a dNTP mix consisting of a final concentration of 0.3 mM dATP, dCTP, dGTP (Sigma-Aldrich) and 0.3 mM dUTP (Bioline Reagents Ltd., London, UK); 1 mM DTT (Invitrogen); 2 U RNase H (Invitrogen) and 50 U E. coli DNA Polymerase I (Invitrogen). Reactions were incubated at 16°C for 2.5 h. The double stranded cDNA was subsequently purified using a QIAquick PCR Purification kit (Qiagen) according to the manufacturer’s instructions and eluted in 30 µl of the provided elution buffer. Blunt-end repair of cDNA was performed in a 100 µl reaction containing 1× T4 DNA ligase buffer with 10 mM dATP (New England Biolabs® Inc., MA, USA), 0.4 mM of each dNTP (Invitrogen), 15 U T4 DNA polymerase (New England Biolabs), 5 U DNA Polymerase I Large [Klenow] Fragment (New England Biolabs) and 50 U T4 polynucleotide kinase (New England Biolabs). Reactions were incubated at 20°C for 30 min and the cDNA was then purified using a QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions and eluted in 32 µl of the provided elution buffer. To facilitate Illumina® GA adaptor ligation, a single ‘A’ base was added to the 3' ends of the blunt-end repaired cDNA samples. 32 µl of purified phosphorylated blunt end-repaired cDNA was included in a final 50 µl reaction mixture containing: 1× Klenow fragment buffer (New England Biolabs); 0.2 mM dATP (Invitrogen), and 15 U Klenow fragment with 3'-to-5' exonuclease activity (New England Biolabs). Reactions were incubated at 37°C for 30 min, after which cDNA was purified using a QIAquick MinElute Kit (Qiagen) according to the manufacturer’s instructions and eluted in 21 µl of the provided elution buffer. Illumina® RNA-seq adaptor ligation reactions (50 µl volumes) involved incubation of 21 µl of phosphorylated blunt-ended cDNA containing a 3'-dATP overhang with 1× Quick DNA ligase buffer (New England Biolabs); 30 nM custom indexed single-read adaptors (see Samples_barcode.xlsx) and 15 U T4 DNA ligase (Invitrogen). Reaction mixes were incubated at room temperature for 15 min and purified using a QIAquick MinElute Kit according to the manufacturer’s instructions (Qiagen) and eluted in 10 µl of the provided elution buffer. Adaptor-ligated cDNA was gel-purified using 2.5% agarose gels stained with 1 μg/ml ethidium bromide (Invitrogen). Gels were electrophoresed at 100 Volts using 1× TAE buffer (Invitrogen) for 75 min at room temperature. Size fractionated bands corresponding to 200 bp (+50 bp) were excised from each sample and purified using a QIAquick Gel Extraction kit (Qiagen) according to the manufacturer’s instructions and eluted in 30 µl of elution buffer. To generate strand-specific RNA-seq libraries, the second strand of the gel-purified adapter-ligated cDNA containing uracil was digested enzymatically in 30 µl reaction volumes containing 1× Uracil-DNA Glycosylase buffer and 1 U Uracil-DNA Glycosylase (Bioline). Reactions were incubated at 37°C for 15 min followed by 94°C for 10 min. PCR enrichment amplifications (50 µl) containing 15 μl of second strand-digested, adaptor-ligated cDNA; 1× Phusion® High-Fidelity DNA polymerase buffer (New England Biolabs); 334 nM each Illumina® PCR primer (Illumina® Inc., San Diego, CA, USA); 0.4 mM each of dATP, dCTP, DGTP and dTTP (Invitrogen) and 1 U Phusion® High-Fidelity DNA polymerase (New England Biolabs). PCR amplification reactions consisted of an initial denaturation step of 98°C for 30 seconds, 18 cycles of 98°C for 10 seconds, 65°C for 30 seconds and 72°C for 30 seconds, followed by a final extension step of 72°C for 5 min. PCR products were visualised following electrophoresis on a 2% agarose gel stained with ethidium bromide (0.6 µg/ml; Invitrogen) and purified to remove PCR-generated adaptor-dimers using an Agencourt AMPure XP kit (Beckman Coulter Genomics, Danvers, MA, USA) according to the manufacturer’s instructions with final elution in 30 μl of 1× TE buffer. All RNA-seq libraries were quantified using a Qubit® Fluorometer and Qubit® double stranded DNA High Sensitivity Assay Kit (Invitrogen). RNA-seq library quality was assessed using an Agilent Bioanalyzer and Agilent High sensitivity DNA chip (Agilent) and confirmed that library insert sizes were ~200-250 bp for all individual libraries. Individual RNA-seq libraries were standardised and pooled in equimolar quantities (10 µM for each individual library). The quantity and quality of the final pooled library was assessed as described above prior to sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Cluster generation and sequencing of the pooled RNA-seq library was carried out on an Illumina® Cluster Station and Illumina® Genome Analyzer IIx sequencer according to the manufacturer’s instructions (Illumina). The pooled library was sequenced as single-end read 84-mers using Illumina® version 4.0 sequencing kits and the standard Illumina® Genome Analyzer IIx pipeline. The Illumina® Sequencing Control Software version 2.9 and Real Time Analysis version 1.9 software packages were used for real-time tracking of the sequencing run, real-time image processing, the generation of base intensity values and base calling.
Sequence reads obtained from seven lanes of the Illumina flow cell were deconvoluted into 14 individual libraries (Raw data files) using the unique indexed barcoded adapters. A Perl script was used to screen adapter artefacts, removing any reads containing a full-length match to the 33 nucleotide Illumina® adapter sequence, allowing up to four mismatches. Read quality was then assessed using FastQC software [version 0.9] (www.bioinformatics.bbsrc.ac.uk/projects/fastqc), revealing low Phred scores at the 3' end of sequence reads. Consequently, nine nucleotides were trimmed from the 3' ends of all sequence reads using the FASTX Toolkit [version 0.0.13] (http://hannonlab.cshl.edu/fastx_toolkit) generating usable reads of 69 nucleotides.
Deconvoluted quality-checked sequence reads were aligned to the B. taurus reference genome (Btau 4.0.63 genome release) with the TopHat splice junction mapper [version 1.3.0], which aligns reads using the Bowtie aligner (version 0.12.7). The Bowtie alignment procedure was configured for strand-specific libraries and to filter only unique hits to the reference genome sequence.
To obtain raw counts per transcript, HTSeq package [version 0.5.3p1] (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html) was used on alignment files in BAM format. HTSeq-count was used with option overlap resolution mode set to intersection non-empty. Counts of uniquely-mapped reads were obtained for all bovine Ensembl genes and transcripts, with separate counts obtained for sense (Processed sense data files) and antisense (Processed antisense data files) DNA strands.
Once raw counts were obtained from HTseq-count, analysis of differential expression was performed in the R statistical programming environment using the edgeR (version 2.2.5) Bioconductor package and lattice [version 0.19-30] (http://lattice.r-forge.r-project.org). First, quality checks were performed by plotting the density of counts per feature (a feature being a gene or transcript generated respectively from the sense or antisense strand) for each sample and also by generating a multidimensional scaling plot of the RNA-seq data. The edgeR software package was used to determine differential expression using the paired-sample statistical test. Filtering of lowly expressed features was performed by retaining only features with at least one count per million in three or more libraries. A normalisation factor was calculated using the default trimmed mean of M values (TMM) method, and the dispersion parameter for each feature was estimated as the Cox-Reid common dispersion method in the edgeR package. Differential expression was evaluated by fitting a negative binomial generalized linear model for each feature and then adjusting the P-value for multiple testing using the Benjamini-Hochberg correction with a false discovery rate (FDR) of 0.05.
Genome_build: Bos taurus reference genome version 4.0.63
Supplementary_files_format_and_content: Tab-delimited text files include raw count values per genes
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| Submission date |
Mar 22, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
David E MacHugh |
| E-mail(s) |
david.machugh@ucd.ie
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| Phone |
+353-1-7166256
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| Organization name |
University College Dublin
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| Department |
College of Agriculture, Food Science and Veterinary Medicine
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| Lab |
Animal Genomics Laboratory
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| Street address |
University College Dublin, Belfield
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| City |
Dublin |
| ZIP/Postal code |
D4 |
| Country |
Ireland |
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| Platform ID |
GPL15750 |
| Series (1) |
| GSE45439 |
Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro |
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| Relations |
| SRA |
SRX254540 |
| BioSample |
SAMN01985921 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1104337_716cn24h_antisense.txt.gz |
84.9 Kb |
(ftp)(http) |
TXT |
| GSM1104337_716cn24h_sense.txt.gz |
94.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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