|
| Status |
Public on Jan 22, 2013 |
| Title |
Map_naive_2h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Intracellular M. ap isolated from infected naïve J774A.1, 2 h post-infection
|
| Organism |
Mycobacterium avium subsp. paratuberculosis K-10 |
| Characteristics |
genotype/variation: Wild-type
host cell: Naïve macrophage
|
| Treatment protocol |
Immediately before infection, M. avium subsp. paratuberculosis culture was pelleted, resuspended in warm RPMI-1640 and passed through a 23-gauge needle five times or until bacterial clumps were dispersed. An approximate 109 CFU bacterial suspension was mixed with 12 ml of RPMI-1640/10% FBS and added to each decanted 15-cm cell culture dish (MOI=50) where J774A.1 cells were grown with or without interferon-gamma treatment. The cells were incubated at 37°C with 5% CO2 for 2 h or 24 h before intracellular bacteria isolation. For the 24 h time point experiments, extracellular bacteria were washed away at 2 h post-infection with 15 ml of warm PBS at least five times or until no visible bacterial particles were observed under an inverted microscope.
|
| Growth protocol |
Mycobacterium avium subsp. paratuberculosis K-10 was grown to OD600=0.5 at 37 degrees and 115 rpm in 7H9 medium (Difco) supplemented with 0.5% glycerol, 0.05% Tween 80, 2 µg/ml mycobactin J (Allied Monitor) and 10% ADC (2% glucose, 5% BSA fraction V, and 0.85% NaCl).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Infected J774A.1 cells were washed with 15 ml ice-cold PBS at least five times or until no visible bacterial particles was observed under an inverted microscope. The washed cells on each dish were then lysed with 10 ml cell lysis buffer (4 M guanidine thiocyanate, 0.5% sodium N-lauryl sarcosine, 25 mM sodium citrate, 0.5% Tween 80 and 0.1 M β-mercaptoethanol) and collected with a rubber cell scrapper. To reduce viscosity and help dissolve cell debris, cell lysats from all dishes were pooled and passed through a 23-gauge needle five times. The lysate was then split into four 14-ml polypropylene centrifuge tubes (Falcon 352059) and centrifuged at 3,200 × g, 4°C for 25 min. Each pellet was washed in 1 ml of TRIzol regent (Invitrogen) twice and then resuspended in 2 ml of TRIzol and split into two 2-ml screw-capped tubes each with 3.0 g of 0.1 mm Zirconia/Silica beads (BioSpect Products) and disrupted in a Mini-BeadBeater-8 (BioSpect Products) at top speed for three pulses of 60 sec with 30 sec intervals on ice. Following a 5-min incubation at room temperature, the supernatant was transferred to RNase-free tubes and centrifuged at 12,000 × g for 15 min. RNA was then isolated according to manufacturer’s instruction. To remove genomic DNA contamination, RNA samples were treated with 10 U of Turbo DNase (Ambion) at 37°C for 30 min. An IS900 241-bp PCR reaction was performed to confirm that no genomic DNA was detectable in the RNA samples. DNase treatments were repeated if needed. Quality of the extracted RNA was examined with a NanoDrop 1000. The ratios of A260/A280 and A260/ A230 must be higher than 1.8 and 1.5, respectively, before proceeding to cDNA synthesis for transcriptome studies.
|
| Label |
Cy3
|
| Label protocol |
Labeling was performed following the standard protocol of NimbleGen Systems Inc., Madison, WI USA.
|
| |
|
| Hybridization protocol |
Three micrograms of the labeled cDNA sample was mixed with NimbleGen hybridization buffer and hybridization component A, covered with a lifted coverslip and hybridized on a NimbleGen microarray chip at 42°C for 16 to 20 hrs. The hybridized chips were washed with NimbleGen wash solutions I, II and III according to Nimblegen's parotocol.
|
| Scan protocol |
Scanning was done with an Axon GenePix 4000B scanner (Molecular Devices) at 5 micron resolution. PMT was adjusted following the suggestions in Nimblegen's protocol.
|
| Description |
Intracellular condition, 2 h in naïve macrophage. Second of two biological replicates used in this condition, each from separate culture
SAMPLE 4
|
| Data processing |
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan 2.2.33 (NimbleGen, Inc.). Intensities on each chip were centered to 1,000 for MeV v4.4.
|
| |
|
| Submission date |
Jan 21, 2013 |
| Last update date |
Jan 22, 2013 |
| Contact name |
CHIA-WEI WU |
| Organization name |
University of Wisconsin-Madison
|
| Department |
Pathobiological Sciences
|
| Street address |
1656 Linden Dr
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL16529 |
| Series (1) |
| GSE43645 |
Expression analysis of Mycobacterium avium subsp. paratuberculosis K-10 within macrophage cell line |
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