The NPRA conditional knockout mouse model (NprAflox/flox) was generated by Shanghai Model Organisms Center, Inc (Shanghai, China). All mice were euthanized by inhaling oxygen with 5% isoflurane at a rate of 1 L/min. The mice were confirmed to be deeply anaesthetized after they were immobile for 1 min. A 25% volume of CO2 gas was constantly flowed into the chamber to bring the mice to the point of clinical death (lack of respiration and heartbeat), and the hearts were subsequently removed to obtain the myocardium.
Extracted molecule
total RNA
Extraction protocol
1-4 ug total RNA was digested with 2uL 5MazF Buffer, 2uL MazF and 0.5uL RNase inhibtor in a 10uL reaction system at 37°C for 15 mins. Another 1ug total RNA was also processed with 2uL 5MazF Buffer,and 0.5uL RNase inhibtor in a 10uL reaction system at 37°C for 15 mins(without MazF), this fraction denoted as Input. The digested RNA were added to 300 uL 1Digestion buffer (50mM Tris-HCl, pH7.4, 150mM NaCl, 0.1% NP40, 40U/uL RNase Inhibitor) containing 2 ug anti-m6A rabbit polyclonal antibody (Synaptic Systems). The reaction was incubated with head-over-tail rotation at 4°C for 2 hours. 20 mL Dynabeads M-280 Sheep Anti-Mouse IgG suspension per sample was blocked with freshly prepared 0.5% BSA at 4°C for 2 hours, washed three times with 300 uL 1Digested buffer, and resuspended in the total RNA-antibody mixture prepared above. The RNA binding to the m6A-antibody beads was carried out with head-over-tail rotation at 4°C for 2 hours. The beads were then washed three times with 500 uL 1Digestion buffer and twice with 500 uL Wash buffer (50 mM Tris-HCl, pH7.4, 50 mM NaCl, 0.1% NP40, 40 U/uL RNase Inhibitor). The enriched RNA was eluted with 200 uL Elution buffer (10 mM Tris-HCl, pH7.4, 1 mM EDTA, 0.05% SDS, 40U Proteinase K, 1uL RNase inhibtor) at 50°C for 1 hour. The RNA was extracted by acid phenol-chloroform and ethanol precipitated.
Label
Cy3
Label protocol
Input and IP enriched MazF digested RNAs(denoted as Modified) were added with equal amount of calibration spike-in control RNA, separately amplified and labeled with Cy3 (for Input) and Cy5 (for Modified) using Arraystar Super RNA Labeling Kit. The synthesized cRNAs were purified by RNeasy Mini Kit. The concentration and specific activity (pmol dye/ug cRNA) were measured with NanoDrop ND-1000.
Hybridization protocol
2.5 ug of Cy3 and Cy5 labeled cRNAs were mixed. The cRNA mixture was fragmented by adding 5 uL 10 Blocking Agent and 1 uL of 25 Fragmentation Buffer, add water to 25uL then heated at 60°C for 30 min, and combined with 25 uL 2 Hybridization buffer. 50 uL hybridization solution was dispensed into the gasket slide and assembled to the m6A Single Nucleotide resolution Microarray slide. The slides were incubated at 60°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 2 of 5
Data processing
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Raw intensities of Modified (IP enriched MazF digested RNA, Cy5-labelled) and Input (total RNA, 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 5 out of 10 samples were retained as All Targets Value in the Excel sheet for further m6A methylation stoichiometry and m6A quantity analyses. m6A methylation stoichiometry was calculated for the percentage of modification based on the Modified (Cy5-labelled) and Input (Cy3-labelled) normalized intensities. m6A quantity was calculated for the m6A methylation amount based on the Modified (Cy5-labelled) normalized intensities. Differentially m6A-methylated RNAs between two comparison groups were identified by filtering with the fold change and statistical significance (p-value) thresholds. Hierarchical Clustering was performed using the R software. GO analysis was performed using topGO package in R environment for statistical computing and graphics, and Pathway analysis was calculated by fisher's exact test.
