RNA Seq of C2C12 cells stimulated with Control, Jag1-expressing or Jag1Ndr-expressing cells. RNA Seq of C2C12 cells stimulated with Control, Jag1-expressing or Jag1Ndr-expressing cells

RNA sequencing of control or Notch1-expressing mouse cells co-cultured with control, Jag1WT, or Jag1Ndr-expressing human cells. Deep sequencing and bioinformatical separation of mouse and human reads reveals transcripts specifically regulated in mouse receptor-expressing cells. Overall design: Mouse C2C12 control and C2C12-FLNotch1, and human HEK-293-Flp-In cells (Hansson et al., 2010): HEK293-Flp control (Flp Ctrl), HEK293-Flp-Jag1WT (Flp Jag1+), HEK293-Flp-Jag1Ndr (Flp Jag1Ndr) were used in this experiment. In one 12-well plate, we seeded 3 wells of mouse C2C12 control cells and 3 wells of C2C12-FLN1 cells, with 3.6x105 cells in 1 mL antibiotic-free medium per well. Cells were allowed to settle for 8 hours. C2C12 control and C2C12-FLN1 cells were transfected with pcDNA5 (1.6 ug/well). All transfections were done using Lipofectamine® 2000 (InvitrogenTM, cat. no. 11668-019) with Opti-MEM® I Reduced Serum Medium (Gibco®, cat. no. 31985-062), according to manufacturer’s instructions. The following day (18 hours post transfection), 3.6x105 cells in 0.5 mL antibiotic-free medium of Flp Ctrl, Flp Jag1+, or Flp Jag1Ndr cells were added. Cells were co-cultured for 6 hours, then lysed in 350 uL per well Buffer RLT (QIAGEN, cat. no. 79216) with 1% 2-Mercaptoethanol (Sigma-Aldrich®, cat. no. M3148) and stored at -80°C until RNA extraction.

本数据集聚焦与对照、Jag1野生型（Jag1WT）或Jag1Ndr表达型人源细胞共培养的对照或Notch1表达型小鼠细胞的RNA测序。通过深度测序与生物信息学方法分离小鼠与人类读段（reads），可解析在小鼠受体表达细胞中特异性受调控的转录本。

实验整体设计：本实验使用小鼠C2C12对照细胞、C2C12-FLNotch1细胞，以及人源HEK-293-Flp-In细胞（Hansson等，2010），具体包括HEK293-Flp对照细胞（Flp Ctrl）、HEK293-Flp-Jag1WT细胞（Flp Jag1+）、HEK293-Flp-Jag1Ndr细胞（Flp Jag1Ndr）。

在一块12孔板中，每孔接种3.6×10^5个处于无抗生素培养基中的小鼠C2C12对照细胞与C2C12-FLN1细胞各3孔，静置8小时待细胞贴壁。随后将pcDNA5（1.6 μg/孔）转染至C2C12对照与C2C12-FLN1细胞，转染操作严格依照制造商说明书，使用Lipofectamine® 2000（InvitrogenTM，货号11668-019）搭配Opti-MEM® I 低血清培养基（Gibco®，货号31985-062）完成。

转染后18小时（即转染次日），向每孔加入0.5 mL无抗生素培养基中悬浮的3.6×10^5个Flp Ctrl、Flp Jag1+或Flp Jag1Ndr细胞。细胞共培养6小时后，每孔加入350 μL含1% β-巯基乙醇（Sigma-Aldrich®，货号M3148）的Buffer RLT裂解液（QIAGEN，货号79216），于-80℃冻存直至后续RNA提取。