RNA-seq of mouse lung tissue in wild type versus BMP9/BMP10 double knockout mice. RNA-seq of mouse lung tissue in wild type versus BMP9/BMP10 double knockout mice

We generated C57BL/6 mice lacking Bmp10 and/or Bmp9 utilizing the Cre-loxP system. Briefly, Bmp9 constitutive deletion resulted from the replacement of exon 2 by a neomycin resistance cassette. Because Bmp10 deletion leads to early embryonic lethality, we used the tamoxifen-inducible Cre system to generate Bmp10-cKO mice (Rosa26-CreERT2;Bmp10lox/lox) by crossing Rosa26-CreERT2 mice with Bmp10lox/lox mice that possess loxP sites flanking exon 2. To generate double-KO (DKO) mice, we crossed these Rosa26-CreERT2;Bmp10lox/lox mice with Bmp9-KO mice. At the age of 8 weeks, mice were treated with tamoxifen (Sigma) by intraperitoneal injection once a day for 5 days at a dosage of 50 mg/kg. At the age of 5 months, Wild Type and DKO mouse lung tissue was flash frozen in liquid nitrogen and stored at -80°C. RNA extraction, RNA sample quality assessment, RNA library preparation, sequencing and raw data analysis were conducted at GENEWIZ, Inc. (South Plainfield, NJ, USA). Total RNA was extracted from frozen tissue using the Qiagen RNeasy Plus Mini kit. RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA). rRNA depletion was performed using Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA). RNA sequencing library preparation used NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 instrument (or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification. After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. Gene counts were calculated from uniquely mapped reads using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. The gene hit counts table was then used for downstream differential expression analysis. A differential gene expression analysis between WT and DKO groups of samples was performed using the R-package DESeq2 (Wald test).

本研究利用Cre-loxP重组酶系统（Cre-loxP system），构建了缺失Bmp10和/或Bmp9的C57BL/6小鼠。具体而言，Bmp9的组成型敲除通过用新霉素抗性基因盒替换其第2外显子实现。由于Bmp10敲除会导致胚胎早期致死，我们采用他莫昔芬诱导型Cre重组酶系统（tamoxifen-inducible Cre system），通过将Rosa26-CreERT2小鼠与携带两侧翼loxP位点的第2外显子的Bmp10lox/lox小鼠杂交，构建了Bmp10条件性敲除（conditional knockout, cKO）小鼠（Rosa26-CreERT2;Bmp10lox/lox）。为构建双敲除（double-KO, DKO）小鼠，我们将上述Rosa26-CreERT2;Bmp10lox/lox小鼠与Bmp9-KO小鼠进行杂交。小鼠于8周龄时，通过腹腔注射给予他莫昔芬（Sigma），每日1次，连续给药5天，给药剂量为50 mg/kg。5月龄时，采集野生型（Wild Type, WT）与DKO小鼠的肺组织，经液氮快速冷冻后于-80℃冰箱保存。RNA提取、RNA样本质量评估、RNA文库构建、测序及原始数据分析均在美国新泽西州南普莱恩菲尔德的GENEWIZ公司完成。采用Qiagen RNeasy Plus Mini试剂盒从冷冻组织中提取总RNA；使用Qubit 2.0 Fluorometer（美国加利福尼亚州卡尔斯巴德的Life Technologies公司）对RNA样本进行定量，并采用Agilent TapeStation（美国加利福尼亚州帕洛阿尔托的Agilent Technologies公司）检测RNA完整性；使用Ribozero rRNA Removal Kit（美国加利福尼亚州圣迭戈的Illumina公司）完成核糖体RNA去除步骤。按照制造商的操作说明，采用NEBNext Ultra RNA Library Prep Kit（美国马萨诸塞州伊普斯维奇的NEB公司）构建Illumina测序用RNA文库。具体步骤如下：首先将富集后的RNA在94℃下片段化15分钟；随后合成第一链与第二链cDNA；对cDNA片段进行末端修复，并在其3’端加腺苷酸尾，之后将通用接头连接至cDNA片段；接着通过有限循环PCR添加索引序列并完成文库富集。采用Agilent Tapestation 4200（美国加利福尼亚州帕洛阿尔托的Agilent Technologies公司）对测序文库进行质检，并通过Qubit 2.0 Fluorometer（美国加利福尼亚州卡尔斯巴德的Invitrogen公司）以及定量PCR（美国加利福尼亚州卡尔斯巴德的Applied Biosystems公司）完成文库定量。将测序文库在流动池的单个泳道上进行簇生成；按照制造商的操作说明，将完成簇生成的流动池加载至Illumina HiSeq 4000测序仪（或同类型设备）中。采用2×150 bp双端（Paired End, PE）测序模式对样本进行测序。图像分析与碱基识别由HiSeq控制软件（HiSeq Control Software, HCS）完成。将Illumina HiSeq测序产生的原始序列数据（.bcl格式文件）通过Illumina bcl2fastq 2.17软件转换为fastq文件并完成去多重化；索引序列识别允许1个碱基错配。在评估原始数据质量后，使用Trimmomatic v.0.36软件对测序读段进行修剪，以去除可能存在的接头序列及低质量碱基。使用STAR比对软件v.2.5.2b将修剪后的读段比对至ENSEMBL数据库发布的小鼠（Mus musculus）GRCm38参考基因组。采用Subread软件包v.1.5.2中的feature Counts工具，从唯一比对的读段中计算基因计数，仅统计位于外显子区域内的唯一比对读段。随后将基因计数表格用于后续的差异表达分析。采用R包DESeq2（Wald检验）对WT与DKO组样本进行差异基因表达分析。