GSM5269155: chrRNA_m6A_IP_Control; Mus musculus; OTHER
MEF cells were fractionated into cytoplasmic and chromatin fractions. Briefly, cells were collected, washed and collected. 200 μL ice cold cytoplasmic lysis buffer (10 mM Tris-HCl pH 7.5, 0.15% NP-40, 150 mM NaCl, 25 mM a-amanitin, 10 U SUPERase. In and protease inhibitor mix) were added to the cell pellet and incubated on ice for 5 min, then gently pipetted up the cell lysate over 2.5 volumes of chilled sucrose buffer (25% sucrose in lysis buffer) and centrifuged at 4 °C with 16,000 g for 10 min. The supernatant was collected as cytoplasmic fraction. Gently added 800 μL ice cold nuclei wash buffer (0.1% Triton X-100, 1 mM EDTA, in PBS, 25 mM a-amanitin, 10 U SUPERase. In and protease inhibitor mix) to the  nuclei pellet and centrifuged at 4 °C with 1,150 g for 2 min. The supernatant was removed completely. The remaining nuclei pellet was resuspended in 200 μL ice cold glycerol buffer (20 mM Tris-HCl pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 50% glycerol,10 U SUPERase. In and protease inhibitor mix) by pipetting up and down, then equal volume of ice cold nuclei lysis buffer (1% NP-40, 20 mM HEPES pH 7.5, 300 mM NaCl, 1M Urea, 0.2 mM EDTA, 1 mM DTT, 25 mM a-amanitin, 10 U SUPERase. In and protease inhibitor mix) was added and mixed by pulse vortexing. After 2 min incubation on ice, the lysis was centrifuged at 4 °C with 18,500 g for 2 min. The supernatant was collected as soluble nuclear fraction/nucleoplasm. Pellet was treated as chromotin-associated fraction. The relavant RNA were extracted by Trizo LS or Trizol. RNA was dephosphorylation for 1 h at 37 °C in a 15 μL reaction (1 × T4 polynucleotide kinase buffer, 10 U SUPERase In and 20 U T4 polynucleotide kinase). The products were separated on a 15% polyacrylamide TBE-urea gel (Invitrogen) and visualized using SYBR Gold (Invitrogen). Selected regions in the gel corresponding to 40–60 nt (were excised. RNA fragments were dissolved by soaking overnight in 400 μL RNA elution buffer (300 mM NaOAc, pH 5.5, 1 mM EDTA, 0.1 U mL−1 SUPERase In). The gel debris was removed using a Spin-X column (Corning), followed by ethanol precipitation. Purified RNA fragments were resuspended in nuclease-free water. Next, 0.15 μg linker (rApp/NNNNCTGTAGGCACCATCAAT/3ddC) was added to the RNA fragments, heated at 70 °C for 90 s and then cooled to room temperature, followed by ligation for 3 h at 22 °C in a 20 μL reaction (T4 RNL2 reaction buffer, 10 U SUPERase In, 15% PEG8000 and 20 U T4 RNA ligase 2 truncated). The reaction was heat inactivated at 80 °C for 10 min and the products were separated on a 10% polyacrylamide TBE-urea gel. Regions in the gel corresponding to 65-85 nt were excised. RNA fragments were dissolved by soaking overnight in 400 μl RNA elution buffer, ethanol purified RNA fragments were re-suspended in nuclease-free water.    For reverse transcription, the following oligos containing barcodes were used (Phos represents phosphorylation, NNN represents random sequence, SpC18 represents Hexa-ethyleneglycol spacer):    (Phos)CTANNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT AGATCTCGGTGGTCGC(SpC18)CACTCA(SpC18)TTCAGACGTGTGCTCTT CCGATCTATTGATGGTGCCTACAG    (Phos)AGCNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT AGATCTCGGTGGTCGC(SpC18)CACTCA(SpC18)TTCAGACGTGTGCTCTT CCGATCTATTGATGGTGCCTACAG    (Phos)ATTNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT    AGATCTCGGTGGTCGC(SpC18)CACTCA(SpC18)TTCAGACGTGTGCTCTT CCGATCTATTGATGGTGCCTACAG    (Phos)CCGNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT AGATCTCGGTGGTCGC(SpC18)CACTCA(SpC18)TTCAGACGTGTGCTCTT CCGATCTATTGATGGTGCCTACAG    The linker ligated RNA sample was mixed with 0.5 mM dNTP and 2.5 mM synthesized RT primer (and incubated at 75 °C for 5 min, followed by incubation on ice for 3 min. The reaction mix was then added with 20 mM Tris-HCl pH 8.4, 50 mM KCl, 5 mM MgCl2, 10 mM DTT, 40 U RNaseOUT and 200 U SuperScript III. Reverse transcription reaction was performed according to the manufacturer's instruction. Reverse transcription products were separated on a 10% polyacrylamide TBE-urea gel. The extended first-strand product band was expected to be approximately 200 nt, and the corresponding region was excised. The cDNA was recovered by using DNA gel elution buffer (300 mM NaCl, 1mM EDTA). First-strand cDNA was circularized in 20 μL of reaction containing 1 × CircLigase buffer, 2.5mM MnCl2, 1 M Betaine and 100 U CircLigase II (Epicenter). Circularization was performed at 60 °C for 1 h, and the reaction was heat inactivated at 80 °C for 10 min, then was precipitated by ethanol.