NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM2884188 Query DataSets for GSM2884188
Status Public on Nov 20, 2018
Title RAW264.7-IL13-24h
Sample type RNA
 
Source name RAW264.7 cells were treated with IL13 (10 ng/mL) for 24 hours
Organism Mus musculus
Characteristics cell line: RAW264.7
treatment: IL13
time: 24h
Treatment protocol RAW264.7 was 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 RAW264.7 cells were cultured in DMEM medium containing 10% FBS and 100U per ml of penicillin-streptomycin in a 5% CO2 humidified incubator at 37°C.
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 microarray 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 microarray 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 temperature. 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 temperature. 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 temperature. 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.
Description To screen for the functional lncRNAs involved in macrophages M2 polarization, we utilized the cellular model of IL13-driven macrophage M2 polarization. RAW264.7 cells were treated with IL13 (10 ng/ml) for 24 hours.
RAW2647 cells were obtained from the Cell Bank of the China Science Academy (Shanghai, China).
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 11, 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 GPL24373
Series (1)
GSE107952 Identifying lncRNA-MM2P as a novel modulator of macrophage M2 polarization

Data table header descriptions
ID_REF
VALUE Log2 transformed quantile-normalized data

Data table
ID_REF VALUE
RB_p_000087108 9.116993678
RB_p_000087109 10.88931382
RB_p_000087110 10.33678548
RB_p_000087111 8.399811959
RB_p_000087112 8.703038389
RB_p_000087113 9.972261849
RB_p_000087114 10.43879185
RB_p_000087115 9.719388821
RB_p_000087116 8.788718333
RB_p_000087117 10.44734157
RB_p_000087118 9.126059116
RB_p_000087119 8.917372079
RB_p_000087120 8.949826711
RB_p_000087121 10.98992663
RB_p_000087122 8.722807531
RB_p_000087123 7.932214752
RB_p_000087124 10.57601187
RB_p_000087125 9.260331519
RB_p_000087126 10.41468524
RB_p_000087127 9.473198384

Total number of rows: 47524

Table truncated, full table size 1247 Kbytes.




Supplementary file Size Download File type/resource
GSM2884188_1-1D.gpr.gz 5.4 Mb (ftp)(http) GPR
Processed data included within Sample table

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap