Illumina HiSeq 2500 sequencing; m6A RNA methylation profiling in hippocampus of POCD mice induced by sevoflurane
All mice were conducted Morris water maze test by EthoVision XT working system (Noldus, Netherlands) as manufacturer's instructions. The escaping latent period of each mouse were recorded. Thirty mice were randomly selected and given 2% sevoflurane for 4 h in an automatic anesthetic chamber with size of 24 cm*12 cm*18 cm (Rurui Technology Co., Ltd, Guangzhou, China). After natural resuscitation, POCD and non-POCD mice were picked up based on the dynamics of escaping latent period. The other twenty mice received normal air were treated as negative control. The mice were sacrificed via cervical dislocation, harvested the hippocampus immediately, washed by cold PBS, and added with RIPA buffer or Trizol Reagent accordingly. The genome-wide m6A was detected by RIP kit (Millipore). RIP Lysis Buffer was added and the products were lysed on ice and collected. Then, the beads were washed with RIP Wash Buffer. After removal of the supernatant, the beads were incubated with 1 μg antibodies of m6A (56593S, CST),for 30 min at room temperature, and normal rabbit immunoglobulin G (Abcam) was used as the control. Subsequently, the cell lysate was incubated with RIP buffer containing magnetic beads. RNA was extracted after proteinase K digestion. Five μg RNA of samples were used for library preparation by NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following manufacturer's instructions.