GSM8257320: OMM 2.3 cells, MA_RIP; 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 μL of UltraPure H2O. C. Add 250 μL of fragmentation buffer to the 50 μL of isolated 2x poly(A) RNA to a final volume of 300 μL. 2. M6A RIP Fragmentation Step of the RIP A. Proceed to the fragmentation of the 300 μL Poly(A) RNA solution at 94°C for exactly 5 minutes using a thermocycler. B. Stop the fragmentation reaction by adding 50 μL of Stop Buffer to a final volume of 350 μL and immediately put on ice. 3. RNA binding to m6A-Dynabeads A. Add 150 μl of pre-equilibrated m6A-Dynabeads (see Notes 2 and 8) to the 350 μl of fragmented RNA from step 2 of section 3.2 to a final volume of 500 μl. B. Allow the fragmented RNA to bind to the m6A-Dynabeads at room temperature while rotating (tail-over-head) at 7 rotations per minute for 1 hour. C. Place the tubes containing the samples on a magnet allowing the bead complexes to cluster until the solution becomes clear. D. Discard the 500 μL liquid phase or supernatant as this fraction represents the m6A negative fragments not captured by the anti-m6A antibody. 4. Washing of m6A-Dynabeads A. Resuspend m6A-Dynabeads-RNA complexes in 500 μL of m6A Binding Buffer, incubate for 3 minutes at room temperature and remove clear supernatant after placing the beads in the magnet. B. Repeat step 1 with 500 μL of Low Salt Buffer. C. Repeat step 1 with 500 μL of High Salt Buffer. Do not exceed 3-minute incubation time for this step to prevent release of the RNA from the beads. D. Repeat step 1 twice with 500 μL of TET buffer. 5. Elution of m6A-positive RNA A. Add 125 μL of 42 °C pre-heated Elution Buffer to the m6A-Dynabead complexes from section 3D, step 4 and incubate at 42 °C for 5 minutes. B. At the end of the 5 minutes, vortex the beads gently and place them on the magnet. C. Collect the liquid phase and transfer to a fresh tube, kept on ice, as it represents the eluate fraction containing the m6A“enriched RNA”. D. Add an additional 125 μL of pre-heated Elution Buffer to the beads and process as described in step 1-3 above for 3 additional times, for a total of 4 elutions. E. Collect the liquid phase obtained at each elution step and pool with the previous ones. Keep sample on ice while working on the next elution. After the fourth round of elution, the final total eluate volume of the m6A positive RNA fraction is 500 μL. 6. Extraction and Cleanup Step of the RIP A. Extract the 500 μl of m6A positive RNA collected in previous step by adding 500 μL of acid phenol-chloroform. B. Centrifuged at 4 °C at 10,000g for 7.5 minutes. C. Carefully collected the upper phase making sure not to touch the inter-phase and transfer to a fresh 1.5 ml tube. D. Add 500 μL of previously tested RNAse free chloroform to the fresh tube, vortex briefly and centrifuged at 4 °C at 10,000g for 7.5 minutes. E. Transfer the upper phase to a fresh 1.5 mL tube and proceed to RNA precipitation (see Note 9) overnight at −20 °C. F. Centrifuged the sample at 4 °C for 20 minutes at 16,000g. G. Wash the pellet twice in 70% ethanol by centrifuging for 10 minutes at 4 °C at 16,000g. H. Dry the pellet at room temperature for 10 minutes prior to re-suspend it in the desired volume (typically 5-6 μl) of Ultra-Pure H20. RIP Library Construction. We have generally utilized 100 ng of RNA (100 ng of input and 100 ng of post m6A-IP positive fraction) for library construction utilizing the Illumina TrueSeq Stranded mRNA. A. Add 13 μL of Fragment, Prime, Finish Mix to the 5 μl m6A positive fragmented RNA obtained (to final volume of 18 μL). B. Centrifuged at 4 °C at 10,000g for 7.5 minutes. Skip the fragmentation step in the Illumina protocol given the RNA has already been fragmented, and proceed immediately to the synthesis of the First Strand cDNA. C. Follow the Illumina protocol to the end. D. Verify the fragment sizes of each individual library on an Agilent BioAnalyzer 2100 or equivalent using High Sensitivity DNA chip. E. Quantify the library by qPCR on using the Kapa library quantification kit according to the manufacturer's instructions. F. Submit the libraries for high-throughput sequencing meRIP-seq