GSM5746929: MS MIIoocyte IP, rep2; Mus musculus; RIP-Seq
Step 1. One-tube rRNA and DNA depletion. To deplete rRNA, we used NEBNext® rRNA Depletion Kit (NEB) according to user manual with minor modifications. Added 3 µl of the RNA/Probe master mix to 12 µl sample. Placed samples in a thermocycler at 95 °C for 2 min and then gradually ramped the temperature (−0.1 °C/s) to 22 °C, held samples at 22 °C for 5 min. Added 5 µl RNase H reaction mix to the samples immediately, incubated samples 37 °C for 30 min and then placed it on ice. Added 30 µl of the DNase I digestion mix to the samples and incubated at 37°C for 30 minutes. Samples were purified with 2.2× volume of RNAClean XP beads. 80% freshly prepared ethanol was used to wash samples twice. The rRNA depleted RNA are eluted from the beads with 78 μl of nuclease-free water (Thermo Fisher Scientific). Added 2 μl RiboLock RNase Inhibitor (40 U/µl) to the samples. Step 2. RNA shearing by sonication. The samples were sonicated for 2 × 30 s using a UP100H Ultrasonic Processor (Hielscher) fitted with a 2-mm probe, using pulse settings with 0.5 s cycles and 27% power. Each sonication cycle is 30 seconds sonication plus 30 seconds on ice. After sonication, 20 µl 5X IP buffer (50 mM Tris-HCl pH 7.5, 750 mM NaCl, 0.5% (vol/vol) NP-40, 5U/µl RiboLock RNase inhibitor) was added into samples. The final volume is 100 µl. Take 10% for regular RNA-seq sequencing as Input control, and the remaining 90% were subjected to Immunoprecipitation. Step 3. Immunoprecipitation and washes. 30 μl Dynabeads (Invitrogen) were washed with 1X IP buffer (200 µl 5× IP buffer with 800 µl nuclease-free water) twice. Added pre-mixed antibody mix (4 µl m6A antibody (Millipore, ABE572), 16 µl 5X IP buffer, 60 µl nuclease-free water) to the washed beads and incubated samples with rotation on the HulaMixer at 4°C for overnight. Pre-incubated antibody-bead complexes were washed in 200 μl 1X IP buffer by vortexing roughly. Performed a second wash by adding 200 μl 1X IP Buffer, vortexed briefly, and aliquoted 10 μl into PCR tubes for each sample, pulse-spined and placed tubes back to magnet until the solution was clear. Supernatants were discarded and added the sonicated RNA mix (100 μl) to antibody-bead complex. Incubated the samples with rotation on the HulaMixer at 4°C for 2 h. After incubation, supernatants were removed. Washed the beads 4 times in the order with the following buffers. First wash with cold medium RIPA Buffer (10 mM Tris–HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% (vol/vol) Triton X-100, 0.2% (vol/vol) SDS, 0.1% (vol/vol) Na-deoxycholate). Two times washes with cold stringent RIPA buffer (10 mM Tris–HCl, pH 8.0, 350 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% (vol/vol) Triton X-100, 0.23% (vol/vol) SDS, 0.1% (vol/vol) Na-deoxycholate), last round wash with cold medium RIPA Buffer. After four times washes, the beads were resuspended with 100 μl 1X IP Buffer to recover for 5 minutes. Placed the samples on magnet and removed the supernatant. Resuspended the pellet in 147.9 μl elution buffer (5 mM Tris–HCl, pH 7.5, 1 mM EDTA, 5 % (vol/vol) SDS, and 1U/µl RiboLock RNase inhibitor). Added 2.1 μL of Proteinase K (NEB) and put samples immediately in a Thermomixer at 1200 rpm, 55°C for 1.5 h. After incubation, briefly spined the tubes and incubated in a Thermomixer at 80°C for 20 min to inactivate the enzyme. After inactivation, placed the samples on a magnet for 2-3 minutes. Transferred the supernatant containing the immunoprecipitated RNA into a new 1.5 ml low-binding tube. Resuspend the beads in 147.9 μl elution Buffer and added 2.1 μL of Proteinase K. Placed immediately in a Thermomixer at 1200 rpm, 55°C for 5 minutes and following inactivated proteinase K in a Thermomixer at 80°C for 20 minutes. Placed back on a magnet for 2-3 minutes. Transferred the supernatant to the previous 1.5 ml low-binding tube to pull both volumes together about 300 μl. Step 4. Ethanol precipitation. Added appropriate volume of nuclease-free water (about 100 μl) to the immunoprecipitated RNA to make final volume is 400 μl. Added 40 μl 3M Sodium Acetate pH 5.2(Thermo Fisher Scientific), 10 μl linear acrylamide 5 mg/μl (Thermo Fisher Scientific), 1000 μl Ice-cold 100% ethanol to the samples in the order. Samples were heavily vortexed and incubated immediately at -80°C for at least 2 h until completely frozen. Centrifuged at 20000 g at 4°C for 15 min and removed the supernatant carefully without disrupting the pellet. Washed the pellet with 1 ml ice-cold 75% ethanol twice and resuspended the pellet in 7 μl nuclease-free water. SMART-Seq® Stranded Kit (Takara) was used to build library according to the manufacturer's protocol with some modification in some steps. Followed the Option 2 protocol without fragmentation for highly fragmented RNA. After PCR1 amplification and AMPure beads purification, added 46.5 µl of nuclease-free water to the beads, skipped the protocol of ribosomal cDNA depletion in Section D, removed the tubes from the magnetic rack and mixed thoroughly by vortexing to resuspend the beads. Incubated at room temperature for 5 min to rehydrate and recovered 46 µl sample. Followed the protocol until sequencing. Libraries were quantified with KAPA Library Quantification Kits (Roche) and the size distribution was checked using TapeStation D1000 ScreenTape (Agilent Technologies). The equally pooled libraries were sequenced using NovaSeq system (Illumina) with 100 bp paired-end mode.