1) RNA extraction and purification.
1. low number of cells or embryos (about 150 mouse oocytes for one reaction) with high viability were washed with BSA/PBS solution (0.5% BSA in 1×PBS) 3 times using mouth pipette under a microscope.
2. Cell or embryo samples were picked through mouth pipette and transfer into 200 μL RNAiso Plus reagent on ice. (Samples can be stored in -80°C for 1 month)
3. 40 μL chloroform was added to the samples.
4. Prepare a MaXtract high density tube and centrifuge at full speed for 5 min to make sure the gel was in the bottom of the tube.
5. The mixture was transferred to a MaXtract high density tube and vortex for 5 seconds to make sure the samples are mixed thoroughly.
6. Centrifuge at full speed for 10 min at 4°C.
7. The aqueous phase was recovered and transferred to a new tube.
8. Add 12μL 3 M NaAc, 2 μL of glycogen (5 mg/mL) to the samples and mixed briefly.
9. Add 120 μL isopropanol to the samples and mixed thoroughly through vortex for 10 seconds.
10. The mixture was incubated at -80°C for 30 min for RNA precipitation.
11. The RNA was precipitated after centrifugation for 15 min at full speed at 4°C.
12. The supernatant was removed carefully to avoid touch the white pellet.
13. Washing twice with 1mL 75% ethanol, centrifuge for 5 min each time.
14. Spin down for another time and remove all the ethanol.
15. Dry the pellet by opening the cap of the tube for several minutes.
14. The pellet was resolved in RNase-free water and mixed thoroughly.
15. 1μL samples can be used to assess the concentration via Qubit RNA HS Assay, or run the Agilent High Sensitivity RNA ScreenTape. (To make sure the extracted RNA are more than 50 ng. RNA with high degradation level would not be recommended.)
2) Antibody-coated bead preparation.
1. 10 μL protein A Dynabeads and 10 μL of protein G Dynabeads was washed twice with 200 μL IP buffer. For each time, adding the IP buffer and mix the beads with pepitte thoroughly, then placing the microcentrifuge tube into a magnetic stand for 1 min and discard the supernatant.
2. Add 200 μL IP buffer to the beads.
3. Add 0.5 μL anti-m6A antibody to the tube and rotate for 2 h in 4°C. (the dose of anti-m6A antibody from different companies need to test before in order to get the highest IP efficiency).
3) RNA fragmentation.
1. Dilute the m6A+ control RNA (GLuc) and m6A- control RNA (CLuc) for 1:1000 using RNase-free water on ice. (control RNA are from Epimark N6-Methyladenosine Enrichment Kit).
2. Prepare the sample mixture on ice:
Sample RNA: 50ng-500ng
diluted m6A+ control RNA (GLuc) : 1μL
diluted m6A- control RNA (CLuc) : 1μL
RNase inhibitor (40 U/μL): 0.5 μL
SUPERase inhibitor (20 U/μL): 0.5 μL
IP buffer without NP-40 : to 50 μL
3. Mixture are transfer into a new microTUBE-50 AFA Fiber Screw-Cap, and
fragmented using a Covaris S220 (peak power: 50, duty factor: 20, cycles/burst: 200, duration: 120 sec).
4) MeRIP and purification.
1. 2.5 μL fragmented RNA mixture was kept as input.
2. Wash the antibody-coated beads twice in 200 μL IP buffer and suspend in 200 μL IP buffer.
3. The remaining fragmented RNA(47.5 μL) was transferred to the antibody-coated beads.
4. Rotated at 20 RPM at 4°C for 4-5 h.
5. Spin briefly and place the tube into a magnetic stand for 1 min. Discard the supernatant and wash the beads with 200μL IP buffer twice, 200μL low-salt buffer twice and 200μL high-salt buffer twice. Each time rotate the tube at 4°C for 5 min.
6. Spin briefly and place the tube into a magnetic stand for 1 min. Discard the supernatant and resuspend the beads with 200 μL of RNAiso Plus reagent. (Stop time point. Samples can be stored in -80°C overnight)
7. The RNA was extracted with the same method as that used for RNA extraction(first part).
8. Elute the RNA with 4μL RNase-free water. The eluted RNA was used for RT-qPCR or library preparation.
5) RT-qPCR
(if the samples are precious，this step can be skipped, and S/N ratio test by qPCR can be done with the library products)
1. Eluted RNA and input RNA was reverse-transcribed using an All-in-One Reverse Transcription Kit directly after ULI-MeRIP. Add the following reagents in a PCR tube:
Eluted RNA and input RNA
5X All in one mix: 4μL
RNase-free water: to 20μL
2. Put the mixture in Thermal cycler, procedure is 25°C 5min, 42°C 50min, 85°C 5min, 4°C ∞
3. cDNA of input and IP samples are used for qPCR to test the IP efficiency.
Calculating the S/N ratio as following equation is important to evaluate the sample quality and whether the experiment is successful.
S/N ratio = 2^(-Ct GLuc or m6A-positive gene in IP + Ct GLuc or m6A-positive gene in input)/2^(-Ct CLuc or m6A-negative gene in IP + CtCLuc or m6A-negative gene in input).
4. Usually we will test the GLuc, CLuc, endogenous gene A(m6A positive genes such like Sox2, c-Myc in ESCs) andGapdh or Actin (m6A-negative genes) to evaluate the S/N ratio. For 50 ng total RNA starting, the S/N ratio of GLuc/CLuc is about 100.
6) Library preparation
1. Library preparation of ULI-MeRIP samples was performed using a SMARTer Stranded Total RNA-Seq Kit
version 2 according to the manufacturer’s protocol.
2. Fragmented input RNA and IP-extracted RNA was reverse-transcribed into cDNA without fragmentation option.
3. The libraries of IP and input samples were amplified for 15-19 cycles and 14-15 cycles, respectively.
4. Quantify the library by Qubit dsDNA HS Assay Kit.
5. Step5)-3 can be done with the library products.