human Ino80 chromatin remodeling complex downstream gene

HeLa cells were cultured in 6-well tissue culture plates (~2x105 cells/well) in DMEM medium containing 10% fetal bovine serum. Arp8 siRNA (sc-60072) was obtained from Santa Cruz, while all specific siRNAs including non-targeting siRNA (D-001206), Ino80 siRNA (D-004176), Ies6 siRNA (D-019327) and Ies2 siRNA (D-009848) SMART pool were from Dharmacon (U.S.A.). The cells were transiently transfected with 10~20 pmol specific siRNAs using Lipofetamine RNAMAX transfection kit (Invitrogen, Cat.No-864425) following the manufacture’s instruction. 24 hours after transfection, cells were divided into new 6-well plates for western blot, RT-PCR, and DNA microarray analysis. 48 hours after siRNAs transfection, cells were harvested and lysed. Whole-cell extract (WCE) were prepared by adding 4 x SDS sample buffer, and total RNA was isolated using TRIzol® LS Reagent (Invitrogen). In addition, cells from 1 well of a 6-well plate were rinsed twice with warm PBS and harvested. Cells were then stored in an RNA hold solution (ER501-01, Beijing Transgen Biotech Co., Ltd.). After the knockdown efficiency was confirmed, cells in RNA hold solution were sent to EMTD Science and Technology Development Co., Ltd. (Beijing, China) for DNA microarray analysis. RNA obtained from knock down four subunits of human INO80 complex in Hela cells and control sample

将HeLa细胞接种于6孔组织培养板中，每孔接种约2×10⁵个细胞，使用含10%胎牛血清的DMEM培养基进行培养。Arp8小干扰RNA（siRNA，货号sc-60072）购自圣克鲁兹（Santa Cruz）公司；其余所有特异性小干扰RNA，包括非靶向siRNA（货号D-001206）、Ino80 siRNA（货号D-004176）、Ies6 siRNA（货号D-019327）以及Ies2 siRNA SMARTpool组合，均购自美国达美康（Dharmacon）公司。使用Lipofectamine RNA MAX转染试剂盒（Invitrogen，货号864425），按照制造商说明书操作，以10~20 pmol的特异性siRNA对细胞进行瞬时转染。转染24小时后，将细胞转接至新的6孔板中，用于蛋白质免疫印迹（Western Blot）、逆转录聚合酶链反应（RT-PCR）以及DNA微阵列分析。siRNA转染48小时后，收集细胞并进行裂解。通过添加4×SDS上样缓冲液制备全细胞提取物（WCE）；总RNA则采用TRIzol® LS试剂（Invitrogen）进行提取。此外，取1块6孔板中1孔的细胞，用预热的PBS洗涤2次后收集，将细胞储存于RNA保护液（ER501-01，北京全式金生物技术有限公司）中。在确认敲低效率后，将保存于RNA保护液中的细胞送至中国北京的EMTD科技发展有限公司，进行DNA微阵列分析。本实验所用RNA取自HeLa细胞中人类INO80复合物4个亚基的敲低样本及对照样本。