Osteoblasts isolated from sp7-GFP transgenics by FACS sorting of 4dpf zebrafish larvae

The transgenic zebrafish line Tg(sp7:sp7-GFP) (ulg071 Tg) was used to obtain fluorescent cells through FACS sorting. Two populations were identifed: P1 displaying low fluorescence, P2 displaying high fluorescence. Cells were collected and sumitted to RNA-Seq A Tg(sp7:sp7-GFP) heterozygous transgenic parent was outcrossed with a wt parent to obtain a clutch of transgenic and non-transgenic offspring. Two populations with low (P1) and high (P2) fluorescence were separated. The collected cells from populations P1 and P2 (about 30-50000 cells) were immediately lysed in 0.5% Triton X-100 containing 2U/µl RNAsin and stored at -80°C. cDNAs were prepared from these lysed cells according to the SMART-Seq 2.0 protocol (Illumina, San Diego, CA, USA) for low input RNA sequencing, while libraries were prepared using the Nextera XT DNA library kit. Third-party re-analysis: Table_P2_Sanger_P1_Sanger_P1_P2.csv (our DESeq2 analysis of P1 and P2 4dpf larvae populations with Sanger dataset from Bioproject PRJEB7244 : ID;gene;baseMean P2_Sanger;log2FoldChange P2_Sanger;padj P2_Sanger;Sanger_baseMean;P2_baseMean;ID;gene;baseMean P1_Sanger;log2FoldChange P1_Sanger;padj P1_Sanger;Sanger_baseMean;P1_baseMean;ID;gene;baseMean;log2FoldChange P1_P2;padj P1_P2;P2_baseMean;P1_baseMean). Table_Counts_P1_P2_Sanger.csv (the counts table complete with the Sanger data).

本研究使用转基因斑马鱼品系Tg(sp7:sp7-GFP)（ulg071 Tg），通过荧光激活细胞分选(Fluorescence-Activated Cell Sorting, FACS)获取荧光标记细胞。共分选得到两个细胞群：荧光强度较低的P1群与荧光强度较高的P2群。收集细胞并进行RNA测序(RNA-Seq)。
将Tg(sp7:sp7-GFP)杂合转基因亲本与野生型(wild type, wt)亲本进行回交，获得一批转基因与非转基因子代，随后分离得到荧光强度分别为低（P1）与高（P2）的两个细胞群。收集的P1与P2群细胞（约30至50000个）立即在含2U/µl RNAsin的0.5% Triton X-100溶液中裂解，并于-80℃保存。根据SMART-Seq 2.0试剂盒（美国加利福尼亚州圣地亚哥Illumina公司）的操作流程，从裂解细胞中制备cDNA以用于低起始量RNA测序；同时使用Nextera XT DNA文库制备试剂盒构建测序文库。
第三方重分析数据集如下：
1. Table_P2_Sanger_P1_Sanger_P1_P2.csv：本研究针对P1与P2群受精后4天(4 days post fertilization, 4dpf)的幼鱼样本，结合来自BioProject数据库PRJEB7244的桑格测序(Sanger sequencing)数据集开展DESeq2分析，该表格字段依次为：ID;gene;baseMean P2_Sanger;log2FoldChange P2_Sanger;padj P2_Sanger;Sanger_baseMean;P2_baseMean;ID;gene;baseMean P1_Sanger;log2FoldChange P1_Sanger;padj P1_Sanger;Sanger_baseMean;P1_baseMean;ID;gene;baseMean;log2FoldChange P1_P2;padj P1_P2;P2_baseMean;P1_baseMean。
2. Table_Counts_P1_P2_Sanger.csv：包含完整桑格测序数据集的计数表。