title: Illumina HiSeq 2500 paired end sequencing; N6-Methyladenosine methylation profile in rat H9C2 cardiomyocytes under the hypoxic stress
design: N6-Methyladenosine methylation profile in rat H9C2 cardiomyocytes under the hypoxic stress
library_construction_protocol: 1×10^7 H9C2 cells were harvested, resuspended in nuclear isolation buffer (1.28 M sucrose, 40 mM Tris pH 7.5, 20 mM MgCl2, 4% Triton X-100) and kept on ice for at least 30 min with frequent mixing. The pellet nuclei were centrifuged with 14,000 rpm for 15 min, resuspended by wash buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 100 U/ml RNAase inhibitor (Solarbio), 1×Protease inhibitors cocktail (Solarbio) and sheared the chromatin through sonication by high power, 5 s on, 30 s off for 30 cycles.  90% nuclei were incubated with 1 μg m6A, NCBP3, METTL3 or eIF4A2 antibodies overnight and 40 μl Protein A/G beads (Thermo Fisher Scientific) 2 hrs by gentle rotation at 4 °C while the rest of 10% were harvested as input. The pellet beads were centrifuged by 3,000 rpm 3 min, washed three times. Both the input and pellet beads were purified by RNAiso plus (Takara, Kusatsu, Japan) and conducted reverse transcription using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The concentration and quality of purified RIP RNAs was measured by Nanodrop 2000 (Thermo Fisher Scientific) and Agilent bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). 4 μg RNA in each group were used for library preparation by NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following manufacturer's instructions.