GSM8307444: Hela-siCtrl-2Days-rp2; Homo sapiens; OTHER
Total RNA was extracted by TRIzol reagent (ThermoFisher Scientific, 15596026) according to the manufacturer's instructions. BS Treatment: 5~10 µL Total RNA, polyA-RNA, or synthesized RNA oligos were mixed with 50 µL BS reagent (prepared by mixing 270 mg Na2SO3 and 34 mg NaHSO3 in 850 μL RNase-free water). Incubate at 70 oC for 3 hours. Then added 95~90 µL water to dilute the sample and used EZ RNA Methylation Kit (Zymo, R5001) for purification. Briefly, added 250 μL RNA Binding buffer and 400 μL EtOH. Mixed well and kept it on ice for 30 min to achieve the best recovery. Then the mixture was transferred to a spin IC column and centrifuged at 12000xg for 1 min. The column was washed with 50 μL RNA wash buffer once. Added 40 μL RNA desulphonation buffer and incubated at room temperature for 1 h. Centrifuged the column at 12000xg for 1 min. The column was washed with 50 μL RNA wash buffer once. BS-treated RNA was eluted with 10 μL water.  Library preparation: 1.5 μg BS-treated/Untreated total RNA were mixed with up-probes and down-probes (30 fmol each). The RNA and probes in total volume of 10 μL were annealed in 1X annealing buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA) by incubating mixture at a temperature gradient: 90 °C for 1 min, 80 °C for 1 min, 70 °C for 1min, 60 °C for 1 min, 50 °C for 1 min, 40 °C for 5 min and hold on 35 °C. Subsequently, a 10 μl of mixture containing 0.8 U Bst 2.0 DNA polymerase (NEB, M0537S), 0.2 nmol dATP in 2X CutSmart buffer was added to the former mixture. The reaction mixture was incubated at 45 °C for 30 min. Then another 10 μL of mixture containing 15% PEG8000, 1.5 nmol ATP, 2.5 U SplintR Ligase (NEB, M0375S) in 1X CutSmart buffer was added and mixed well. The whole mixture was incubated at 25 °C for 20 min. 20 μL water was added to dilute the reaction mixture. Ligated product was purified by Zymo RNA Clean & Concentrator-5 and eluted with 15 μL water. The eluted product was added with 7.5 μL water, 2.5 μL Unique Dual Index (NEB, E6442S) and 25 μL LongAmp Taq 2X Master Mix (2X) (NEB, M0287S). PCR was run at the following condition: 94 °C, 30 s; (94 °C, 15 s; 62 °C, 30 s; 70 °C, 15 s)×C cycles (C=12 for Untreated samples, C=14 for BS-treated samples); 70 °C, 5 min; 4 °C, hold-on. PCR product was purified by running 2.5% agarose TBE gel and recovered by Qiagen MinElute Gel Extraction Kit. The libraries were pooled in equal molarities and sequenced on one lane of an Illumina NextSeq 2000 platform. Around 0.5 M reads were acquired for each library.