C3_combined_R2.fastq.gz

Glucocorticoid steroid hormones play essential roles for maturation and growth of many fetal organs including the lung and heart, yet the kidney-specific roles are not well characterised. Glucocorticoids activate the intracellular glucocorticoid receptor (GR) that acts primarily as nuclear transcriptional regulators. We analysed the effect of loss of GR expression on the fetal kidney transcriptome at E18.5 by RNA sequencing. Total RNA was extracted from control (n=4) and GR-null (n=3) kidneys. Loss of GR expression resulted in 2473 differentially expressed genes (FDR < 0.05), 288 genes with absolute LogFC > 1 & FDR < 0.05, which identified 16 upregulated and 25 downregulated primary ciliary genes (FDR < 0.05). Primary cilia are cell signalling and environment sensing organelles that protrude from cell membranes and play important roles during embryogenesis and tissue homeostasis. Little is known of the cellular pathways regulating ciliogenesis. Our findings indicate a role of glucocorticoid signalling in primary cilia formation in renal tubular cells of the developing mouse kidney.Total RNA was isolated from embryonic kidneys using TRIzolTM reagent (Invitogen, USA) according to the manufacturer’s instructions. Total RNA was analysed using a Bioanalyzer 2100 (Agilent Technologies, USA) and Next generation RNA sequencing (NGS RNA-seq) was performed by Genewiz Biotechnology, Suzhou, China. RNA sequencing (20 million reads) was performed on the Illumina Hiseq platform, in a 2 x 150 bp paired-end format.Total RNA of each sample was extracted using TRIzol reagent (Invitrogen) following the manufacturer's instructions.Next generation sequencing library preparations were constructed according to the manufacture's protcol. The The poly(A) mRNA isolation was performed using Poly(A) mRNA Magnetic Isolation Module or rRNA removal Kit. The mRNA fragmentation and priming was performed using First Strand Synthesis Reaction Buffer and Random Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified double stranded cDNA by beads was then treated with End Prep Enzyme Mix to repair both ends and add a dA tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor ligated DNA was then performed using beads, and fragments of ~420 bp (with the approximate insert size of 300 bp) were recovered. Each sample was then amplified by PCR for 13 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using beads, validated using an Qsep100 (Bioptic, Taiwan, China), and quantified by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).In order to remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format were processed by Cutadapt (V1.9.1) to be high quality clean data.Firstly, reference genome sequences and gene model annotation files of relative species (GRm39.97) were downloaded from genome website, such as UCSC, NCBI, ENSEMBL. Secondly, Hisat2 (v2.0.1) was used to index reference genome sequence. Finally, clean data were aligned to reference genome via software Hisat2 (v2.0.1).In the beginning transcripts in fasta format are converted from known gff annotation file and indexed properly. Then, with the file as a reference gene file, HTSeq (v0.6.1) estimated gene and isoform expression levels from the pair-end clean data.

糖皮质类固醇激素对包括肺、心脏在内的多种胎儿器官的成熟与生长发挥关键作用，但其在肾脏中的特异性功能尚未得到充分阐明。糖皮质激素可激活细胞内的糖皮质激素受体（glucocorticoid receptor, GR），该受体主要作为核转录调控因子行使功能。本研究通过RNA测序技术，分析了胚胎发育至E18.5时GR表达缺失对胎肾转录组的影响。我们从对照组（n=4）和GR敲除组（n=3）的肾脏组织中提取总RNA。GR表达缺失导致2473个差异表达基因（错误发现率（False Discovery Rate, FDR）< 0.05），其中288个基因的绝对对数倍变化（logarithmic fold change, LogFC）>1且FDR < 0.05；进一步分析鉴定出16个上调、25个下调的初级纤毛基因（FDR < 0.05）。初级纤毛是一类从细胞膜向外突出的细胞信号传导与环境感知细胞器，在胚胎发生及组织稳态维持过程中发挥重要作用。目前对于调控纤毛发生的细胞通路的了解仍十分有限。本研究结果表明，糖皮质激素信号通路在发育中小鼠肾脏的肾小管细胞初级纤毛形成过程中具有重要作用。我们按照制造商说明书，使用TRIzol™试剂（Invitogen，美国）从胚胎肾脏中分离总RNA。采用生物分析仪2100（Agilent Technologies，美国）对总RNA进行质量分析，下一代RNA测序（NGS RNA-seq）由中国苏州吉凯基因生物技术有限公司完成。测序在Illumina Hiseq平台上进行，采用2×150 bp双端测序模式，每个样本产出约2000万条reads。每个样本的总RNA均按照制造商说明书，使用TRIzol试剂（Invitrogen）进行提取。下一代测序文库的构建参照制造商的实验流程进行：首先采用Poly(A) mRNA磁珠分离试剂盒或核糖体RNA去除试剂盒完成Poly(A) mRNA的富集；随后使用第一链合成反应缓冲液与随机引物完成mRNA的片段化与引物结合；利用ProtoScript II逆转录酶合成第一链cDNA，再通过第二链合成酶混合物合成第二链cDNA。经磁珠纯化的双链cDNA随后通过末端修复酶混合物进行末端修复并添加dA尾，随后通过T-A连接反应在两端添加测序接头。之后使用磁珠对连接有测序接头的DNA进行片段筛选，回收约420 bp的片段（插入片段大小约为300 bp）。每个样本随后使用P5和P7引物进行13轮PCR扩增，两种引物均带有可与测序流动槽互补以进行桥式PCR的序列，其中P7引物带有6碱基的索引序列以实现多重测序。PCR产物经磁珠纯化后，使用Qsep100分析仪（Bioptic，中国台湾）进行质量验证，并通过Qubit 3.0荧光计（Invitrogen，美国加利福尼亚州卡尔斯巴德）进行定量。为去除测序接头、聚合酶链式反应（polymerase chain reaction, PCR）引物等技术序列，并过滤掉质量值低于20的碱基，我们使用Cutadapt（版本1.9.1）对fastq格式的原始过滤数据进行处理，得到高质量的清洁数据。首先，从UCSC（加州大学圣克鲁兹分校基因组浏览器）、NCBI（美国国家生物技术信息中心）、ENSEMBL（欧洲生物信息研究所基因组数据库）等基因组数据库下载对应物种的参考基因组序列及基因模型注释文件（GRm39.97）；其次，使用Hisat2（版本2.0.1）对参考基因组序列构建索引；最后，通过Hisat2（版本2.0.1）软件将清洁数据比对至参考基因组。首先将已知的gff注释文件转换为fasta格式的转录本序列并完成索引构建；随后以该文件作为参考基因文件，利用HTSeq（版本0.6.1）对双端清洁数据中的基因及转录本表达水平进行定量。