|
| Status |
Public on Nov 20, 2018 |
| Title |
BMDMs-Control |
| Sample type |
RNA |
| |
|
| Source name |
BMDMs were untreated
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
age: 6 weeks
cell type: bone marrow-derived macrophages (BMDMs)
treatment: none
time: 0h
|
| Treatment protocol |
BMDMs were seeded in 6-well plates at a density of 2×10^5/well and stimulated with 10 ng/ml IL13 for indicated times.
|
| Growth protocol |
Six-week-old C57BL/6 mice were sacrificed and soaked in 75% ethanol. Bone marrow cells were cultured in DMEM containing 10% FBS and 50 ng/ml M-CSF for three days to obtain BMDMs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1. For proper amount of cells, add 1mL of TRIzol reagent and mix by briefly vortex. Stand for 5 mins at room temperature. If a lot of insoluble material exists after homogenization and room-temperature incubation, remove by centrifugation at 12,000Xg for 10 mins at 4ºC. Transfer the supernatant to a fresh tube. 2. Add 200ul chloroform for 1ml mixture and vortex for 15 sec. Then stand for 5 mins at room temperature. 3. Centrifuge the mixture for 15 min at ≥12000xg at 4ºC. 4. Transfer the aqueous phase (the upper layer containing RNA) to a fresh tube. 5. Add equal volume of 100% Isopropanol to the aqueous phase, mix by inverting the tube several times. 6. Incubate at ‐20ºC for ≥30 min. 7. Centrifuge at 13000 rpm for 15 min at 4ºC. 8. Wash the pellet with 80% EtOH, invert the tube several times. Centrifuge at 7500 rpm for 5 min at -4ºC. 9. Carefully discard the supernatant without touching the pellet, dry the pellet at room temperature. 10. Dissolve the pellet with 20‐30ul DEPC-treated H2O, based on the size of the pellet. RNA solution should be stored at -80ºC.
|
| Label |
Cy5
|
| Label protocol |
The platinum complex of ULS molecule was used to form coordinative bound with the N7 position of guanine. Labeling efficiency can be calculated by the concentration of CyDye and RNA measured by K5500 micro-spectrophotometer. For good microarray result, labeling efficiency should be between 1.0~3.6.
|
| |
|
| Hybridization protocol |
Pre-hybridization: 1) Assembled CustomArray™ microarray with Hybridization Cap and Clips. 2) Fill the hybridization chambers with nuclease-free water. Avoid introducing air bubbles into the chambers. Cover the solution portals with adhesive tape to evaporation. 3) Incubate at 65°C for 10min. 4) Remove the microarray from the incubator and bring to room temperature. Remove the adhesive tape and aspirate the water out of the hybridization chambers. 5) Fill the hybridization chambers with Pre-hybridization Solution.Mix gently by pipetting. A small air bubble can be introduced to improve the mixing process if the arrays are rotated. Wipe the surface clean with a lint-free tissue and cover the solution portals with adhesive tape. 6) Load the microarry onto the rotisserie in the hybridization oven and incubate at 37°C for 60 min with gentle rotation.
Hybridization: 1) Prepare the hybridization solution with labeled RNA target. 2) Denature the hybridization solution at 95°C for 3 minutes, and then cool for 20 seconds on ice. Remove the hybridization solution from ice and keep it at room temperature. 3) Spin down the hybridization solution in a microcentrifuge for 5 seconds at maximum speed. 4) Remove the adhesive tape and pipet the pre-hybridization solution out of the hybridization chambers. 5) Fill the hybridization chambers with the hybridization solution and mix gently with repeated pipetting. A small air bubble will form, and it will improve the mixing process if the arrays are rotated in the hybridization rotisserie oven. 6) Carefully wipe excess solution from the surface of the hybridization cap with a lint-free tissue, and cover the solution portals with adhesive tape. 7) Load the microarry onto the rotisserie in the hybridization oven and incubate at 37°C for 16 hours with gentle rotation.
Hybridization washing: 1) Remove the microarray from the hybridization oven. Remove the adhesive tape and pipet the hybridization solution out of the chambers. 2) Using the Wash solution 1, rinse the hybridization chambers, fill the chambers, and incubate the array for 3 minutes at room temperatrue. Remove the Wash solution 1 from the hybridization chambers. 3) Using the Wash solution 2, rinse the hybridization chambers, fill the chambers, and incubate the array for 3 minutes at room temperatrue. Remove the Wash solution 2 from the hybridization chambers. 4) Using the Wash solution 3, rinse the hybridization chambers, fill the chambers, and incubate the array for 3 minutes at room temperatrue. Remove the Wash solution 3 from the hybridization chambers. 5) Repeat step one more time for a total of two washings with the Wash solution 3.
|
| Scan protocol |
Imaging is done by laser scanner GenePix4000B (Molecular Device). At first, remove the microarray from the clamp and place it horizontally. Then cover the semiconductor microarray surface with the imaging solution. Using a fresh lifterslip cover it, taking care not to introduce air bubbles. At last, load the microarray into the scanner to scan.
|
| Data processing |
The raw signal is normalized with quantile normalization method. Standard selection criteria to identify differentially expressed genes are established at P < 0.05.
|
| |
|
| Submission date |
Dec 12, 2017 |
| Last update date |
Nov 21, 2018 |
| Contact name |
Rong Dong |
| E-mail(s) |
11319042@zju.edu.cn
|
| Organization name |
Zhejiang University
|
| Department |
College of Pharmaceutical Sciences
|
| Street address |
No.886 Yuhangtang Road
|
| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310000 |
| Country |
China |
| |
|
| Platform ID |
GPL24378 |
| Series (1) |
| GSE107979 |
Identifying lncRNAs as novel modulators of macrophage M2 polarization |
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