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Sample GSM7998003 Query DataSets for GSM7998003
Status Public on May 21, 2024
Title liver tissues from the healthy donors 3
Sample type RNA
 
Channel 1
Source name liver tissue
Organism Homo sapiens
Characteristics donor sex: male
donor age: 75y
Treatment protocol No specific treatment was performed.
Growth protocol The liver tissues of a 3mm×3mm from different donors were collected during normal surgical operation associated with their symptomatic treatment. And the samples were kept at 4 °C.
Extracted molecule total RNA
Extraction protocol The total RNA were extracted by TRIzol reagent (CWbio, China), using the standard protocol for trizol extract. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label Cy5
Label protocol The total RNAs were immunoprecipitatedwith anti-N6-methyadenosine (m6A) antibody. The modified RNAs were eluted from the immunoprecipitated magnetic beads as the “IP”. The unmodified RNAs were recovered from the supernatant as “Sup”. The “IP” and “Sup” RNAs were labeled with Cy5 and Cy3 respectively as cRNAs in separate reactionsusing Arraystar Super RNA Labeling Kit. Reagents:Affinity purified anti-m6A rabbit polyclonal antibody (Synaptic Systems, 202003);DynabeadsTM M-280 Sheep Anti-Rabbit IgG (Invitrogen, 11203D);RNase inhibitor (Enzymatics, Y9240L)
 
Channel 2
Source name liver tissue
Organism Homo sapiens
Characteristics donor sex: male
donor age: 75y
Treatment protocol No specific treatment was performed.
Growth protocol The liver tissues of a 3mm×3mm from different donors were collected during normal surgical operation associated with their symptomatic treatment. And the samples were kept at 4 °C.
Extracted molecule total RNA
Extraction protocol The total RNA were extracted by TRIzol reagent (CWbio, China), using the standard protocol for trizol extract. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label Cy3
Label protocol The total RNAs were immunoprecipitatedwith anti-N6-methyadenosine (m6A) antibody. The modified RNAs were eluted from the immunoprecipitated magnetic beads as the “IP”. The unmodified RNAs were recovered from the supernatant as “Sup”. The “IP” and “Sup” RNAs were labeled with Cy5 and Cy3 respectively as cRNAs in separate reactionsusing Arraystar Super RNA Labeling Kit. Reagents:Affinity purified anti-m6A rabbit polyclonal antibody (Synaptic Systems, 202003);DynabeadsTM M-280 Sheep Anti-Rabbit IgG (Invitrogen, 11203D);RNase inhibitor (Enzymatics, Y9240L)
 
 
Hybridization protocol 2.5 μg of Cy3 and Cy5 labeled cRNAs were mixed. The cRNA mixture was fragmented by adding 5 μL 10 Blocking Agent and 1 μL of 25 Fragmentation Buffer, heated at 60°C for 30 min, and combined with 25 μL 2 Hybridization buffer. 50 μL hybridization solution was dispensed into the gasket slide and assembled to the m6A-mRNA&lncRNA Epitranscriptomic Microarray slide. The slides were incubated at 65°C for 17 hours in an Agilent Hybridization Oven.
Scan protocol The hybridized arrays were washed, fixed and scanned using an Agilent Scanner G2505C.
Description Biological replicate 3 of 3. liver tissue from the healthy donor.
Data processing Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images.
Raw intensities of IP (immunoprecipitated, Cy5-labelled) and Sup (supernatant, Cy3-labelled) were normalized with average of log2-scaled Spike-in RNA intensities. After Spike-in normalization, the probe signals having Present (P) or Marginal (M) QC flags in at least 3 out of 9samples were retained for further “m6A methylation level” and “m6A quantity” and “expression level”analyses. “m6A methylation level” was calculated for the percentage of modification based on the IP (Cy5-labelled) and Sup (Cy3-labelled) normalized intensities. “m6A quantity” was calculated for the m6A methylation amount based on the IP (Cy5-labelled) normalized intensities. “RNA expression level” was calculatedbased on the total of IP (Cy5-labelled) and Sup (Cy3-labelled) normalized intensities, and an additional quantile normalization method of limma package was used to normalize the RNA expression level between arrays before probes flag screening.Differentially m6A-methylated RNAs or expressed between two comparison groups were identified by filtering with the fold change and statistical significance (p-value) thresholds.
 
Submission date Dec 29, 2023
Last update date May 21, 2024
Contact name Chao Fan
E-mail(s) superfancity@hotmail.com
Organization name The Fourth Military Medical University
Street address #169, Changle Road, Xincheng District
City Xi'an
ZIP/Postal code 710032
Country China
 
Platform ID GPL25759
Series (1)
GSE252249 m6A-mRNA&lncRNA Epitranscriptomic Microarray analysis for human liver tissues of healthy, HBV-infected with and without cirrhosis donors.

Supplementary file Size Download File type/resource
GSM7998003_L13.txt.gz 5.9 Mb (ftp)(http) TXT
Raw data provided as supplementary file
Processed data are available on Series record

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