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| Status |
Public on Aug 10, 2020 |
| Title |
Liver_6weeks_TH1_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Liver, 6weeks, TH1, replicate1
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
tissue: Liver
Sex: male
age: 14weeks
feeding treatment: fed with Klebsiella pneumoniae strain TH1
weeks post gavage: 6
|
| Treatment protocol |
After the mice were executed, the liver was taken and the liquid nitrogen was quickly frozen
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Incubate at 37°C for 30 minutes.Add 350μl Buffer RLT, and mix well.Add 250μl ethanol (96–100%) to the diluted RNA, and mix well by pipetting. Do not centrifuge. Proceed immediately to step 5.Transfer the sample (700μl) to an RNeasy Mini spin column placed in a 2ml collection tube. Close the lid gently, and centrifuge for 15s at ≥8000 x g (≥10,000 rpm).Discard the flow-through.Add 500μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15s at≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through. Add 500μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2min at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Place the RNasy spin column in a new 2ml collection tube, and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1min. Place the RNeasy spin column in a new 1.5ml collection tube. Add appropriate RNase-free water (please see report “RNA-QC”) directly to the spin column membrane. Close the lid gently, and centrifuge for 1min at ≥8000 x g (≥10,000 rpm) to elute the RNA. RNA quantification and quality control (Passed-please see report “RNA-QC”)
|
| Label |
Cy3
|
| Label protocol |
Add 200ng of total RNA (2.5μL) to a 1.5mL microcentrifuge tube. Add 0.8 μL of T7 Promoter Primer. Add 2 μL of Spike Mix. Denature the primer and the template by incubating the reaction at 65°C in a circulating water bath for 10 minutes. Place the reactions on ice and incubate for 5 minutes. Immediately prior to use, gently mix the components listed in the following table for the cDNA Master Mix by adding in the order indicated, and put the reaction solution on ice.Briefly spin each sample tube in a microcentrifuge to drive down the contents from the tube walls and the lid. Return the tubes to ice. Add 4.7μL of cDNA Master Mix to each sample tube and mix by pipetting up and down. Incubate samples at 40°C in a circulating water bath for 2 hours. Move samples to a 70°C circulating water bath and incubate for 15 minutes. Move samples to ice. Incubate for 5 minutes. Spin samples briefly in a microcentrifuge to drive down tube contents from the tube walls and lid. Immediately prior to use, gently mix the components listed in the following table in the order indicated for the Transcription Master Mix by pipetting at room temperature. Add 6μL of Transcription Master Mix to each sample tube. Gently mix by pipetting. Incubate samples in a circulating water bath at 40°C for 2 hours.
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| |
|
| Hybridization protocol |
Add 500μL of nuclease-free water to the vial containing lyophilized 10X Blocking Agent. Mix by gently vortexing. Equilibrate water bath to 60°C. For each microarray, add each of the components as indicated in the tables as below to a 1.5mL nuclease-free microfuge tube. Mix well but gently on a vortex mixer. Incubate at 60°C for exactly 30 minutes to fragment RNA. Add 2x GEx Hybridization Buffer HI-RPM to the array to stop the fragmentation reaction. Mix well by careful pipetting. Take care to avoid introducing bubbles. Do not mix on a vortex mixer; mixing on a vortex mixer introduces bubbles. Spin for 1 minute at room temperature at 13,000 rpm in a microcentrifuge to drive the sample off the walls and lid and to aid in bubble reduction. Place sample on ice and load onto the array as soon as possible. Load a clean gasket slide into the Agilent SureHyb chamber base with the label facing up and aligned with the rectangular section of the chamber base. Ensure that the gasket slide is flush with the chamber base and is not ajar. Slowly dispense the volume of hybridization sample onto the gasket well in a “drag and dispense” manner. Slowly place an array “active side” down onto the SureHyb gasket slide, so that the “Agilent”-labeled barcode is facing down and the numeric barcode is facing up. Verify that the sandwich-pair is properly aligned. Place the SureHyb chamber cover onto the sandwiched slides and slide the clamp assembly onto both pieces. Hand-tighten the clamp onto the chamber. Vertically rotate the assembled chamber to wet the gasket and assess the mobility of the bubbles. Place assembled slide chamber in rotisserie in a hybridization oven set to 65°C. Set your hybridization rotator to rotate at 10rpm. Hybridize at 65°C for 17 hours.
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| Scan protocol |
Assemble the slides into an slide holder. Place assembled slide holders into scanner carousel. Verify scan settings for one-color scans. Click Scan Slot m-n on the Scan Control main window where the letter m represents the Start slot where the first slide is located and the letter n represents the End slot where the last slide is located.
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| Description |
Gene expression after 6weeks TH1-fed
C3T_6weeks
|
| Data processing |
Open the Agilent Feature Extraction (FE) software.Add the images (.tif) to be extracted to the FE Project. Set FE Project Properties. Check the Extraction Set Configuration. Save the FE Project (.fep) by selecting File > Save As and browse for desired location. Select Project > Start Extracting and export data to txt.
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| |
|
| Submission date |
Aug 10, 2017 |
| Last update date |
Aug 10, 2020 |
| Contact name |
Jing Lu |
| E-mail(s) |
13716207513@163.com
|
| Organization name |
Academy of Military Medical Sciences
|
| Street address |
Beijing, Fengtai District,100071, China
|
| City |
Beijing |
| ZIP/Postal code |
100071 |
| Country |
China |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE102489 |
Fatty liver disease caused by high alcohol-producing Klebsiella pneumoniae |
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