GSM8257321: OMM 2.3 cells, NC_input; Homo sapiens; RIP-Seq
A. Crush frozen tissue sample using the chilled mortar and pestle，and grind tissue sample for 5 minutes until sample becomes powdery. B. For 50-100mg of fresh weight of sample, add 1 mL of TRIzol to sample and mix vigorously. C. Add 200 ul of chloroform, and shake by hand for (at least) 15 seconds with increasing speed from slow to fast to vigorous. Incubate at room temperature for 5 minutes. D. Centrifuge for 15 minutes at 13000 rpm at 4° C.. E. Transfer the aqueous phase to a sterile 1.5 mL centrifuge tube. Add equal volume of 100% isopropanol to the aqueous phase, and incubate at room temperature for 10 minutes. F. Centrifuge for 15 minutes at 13000 rpm at 4° C, and remove the supernatant. G. Add 1 mL of cold 75% ethanol to wash the pellet. H. Centrifuge for 5 minutes at 13000 rpm at 4° C. Discard the wash, and dry the RNA pellet for 5–10 minutes. I. Dissolve RNA in RNase-free water, and incubating for 10 minutes at 55 to 60°C. 1. M6A RIP A. Each biological replicate for m6A-seq starts by using 400 μg of total RNA yielding approximately 10 μg of double poly(A) selected RNA. B. Resuspend each sample obtained from step 1 (10 μg of poly(A) RNA) in 50 μ