Akap11 is a positive regulator of osteogenesis in mouse osteoblastic cell line MC3T3-E1

Purpose: This is a post-GWAS functional study that aims to determine the effect of Akap11 knockout in mouse preosteoblastic cell line MC3T3-E1 during osteogenesis. Methods: mRNA profile of WT (treated with CRISPR and remains unedited) and Akap11 knockout MC3T3-E1 cells were generated by RNAseq from pooled RNA (from 3 biological triplicate at equal quantity) of various timepoint during osteogenesis (Day 0, 7,16, 25).100ng total RNA was used for library construction by KAPA Stranded mRNA-Seq Kit (Roche). Pair-End 101bp sequencing was performed in the Illumin HiSq1500 sequencer using the HiSeq SBS Kit v4 (Illumina). Data was collected with SCS version 1.4.8. Base calling was performed using Illumina's RTA (Version 1.12.4.2). 7 samples were sequenced per lane and achieved a sequencing depth of ~65 million read per sample. Results: Expression of osteogentic markers in Akap11 knockout MC3T3-E1 cells were suppressed. Genes invovled in IGF-I signaling pathway were signficantly altered. Overall design: mRNA profile of Day0, 7, 16, 25 WT (treated with CRISPR and remains unedited) and Akap11 knockout MC3T3-E1 cells were generated by pooled PolyA mRNA Sequencing .

研究目的：本研究为全基因组关联分析（Genome-Wide Association Study, GWAS）后的功能学研究，旨在明确Akap11基因敲除对小鼠成骨前体细胞系MC3T3-E1成骨过程的影响。
研究方法：针对经CRISPR处理但未发生编辑的野生型与Akap11基因敲除的MC3T3-E1细胞，在成骨诱导的第0、7、16、25天收集样本，将3份生物学重复的等量混合RNA进行RNA测序，以获取mRNA表达谱。实验采用Roche公司的KAPA链特异性mRNA测序建库试剂盒，以100ng总RNA完成文库构建；使用Illumina HiSeq 1500测序仪及HiSeq SBS Kit v4（Illumina）开展101bp双端测序；测序数据通过SCS v1.4.8版本软件采集，碱基识别步骤采用Illumina RTA软件（版本1.12.4.2）完成。每个测序泳道包含7个样本，单样本测序深度约为6500万条reads。
研究结果：Akap11基因敲除的MC3T3-E1细胞中成骨标志物的表达受到抑制，胰岛素样生长因子-I（Insulin-like Growth Factor-I, IGF-I）信号通路相关基因发生显著表达改变。
整体实验设计：对成骨诱导第0、7、16、25天的野生型（经CRISPR处理但未发生编辑）与Akap11基因敲除MC3T3-E1细胞，通过混合PolyA mRNA测序获取其mRNA表达谱。