RNA Sequencing based identification of osteoclast-specific genes in presence of plumbagin

Bone marrow-derived osteoclast precursors were obtained from femora and tibiae of balb/c mice. The mouse osteoclast precursors were seeded in 6 well plate with complete alpha-MEM and incubated with M-CSF overnight. The next day, cells were treated with or without RANKL in the absence or presence of plumbagin for 24, 48, 72 and 96 hours. After the respective time treatment, cells were stored in Trizol and RNA was isolated and quantitated using NanoDrop 1000 spectrophotometer. The RNA sequencing were carried out the Illumina HiSeq4000 system.

本研究所用骨髓源性破骨细胞前体（bone marrow-derived osteoclast precursors）提取自BALB/c小鼠的股骨与胫骨。将该前体细胞接种于搭载完全α-MEM培养基的6孔板中，加入巨噬细胞集落刺激因子（M-CSF）后过夜培养。次日，分别在有或无斑鸠菊素（plumbagin）的条件下，以核因子κB受体活化因子配体（RANKL）处理细胞，处理时长分别设置为24、48、72及96小时。经对应时长的干预后，将细胞留存于Trizol试剂内，随后提取总RNA并通过NanoDrop 1000分光光度计完成RNA定量。采用Illumina HiSeq4000测序系统开展RNA测序实验。