C3_combined_R1.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.

糖皮质类固醇激素在包括肺、心脏在内的多种胎儿器官的成熟与生长过程中发挥核心作用，但其肾脏特异性功能尚未得到充分表征。糖皮质激素可激活胞内糖皮质激素受体（glucocorticoid receptor, GR），该受体主要作为核转录调控因子发挥功能。本研究通过RNA测序技术，分析了胚胎发育第18.5天（E18.5）胎鼠肾脏中GR表达缺失对转录组的影响。实验从对照组（n=4）及GR基因敲除（GR-null，n=3）的肾脏组织中提取总RNA。结果显示，GR表达缺失导致2473个差异表达基因（错误发现率<0.05，False Discovery Rate, FDR），其中288个基因的绝对对数倍变化（log2 fold change, LogFC）>1且FDR<0.05；进一步筛选得到16个上调、25个下调的初级纤毛（primary cilia）相关基因（FDR<0.05）。

初级纤毛（primary cilia）是一类从细胞膜伸出的细胞信号与环境感知细胞器，在胚胎发生及组织稳态维持中发挥关键作用。目前对于调控纤毛发生的细胞通路尚缺乏深入认知。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调控作用。

采用TRIzol™试剂（Invitogen，美国），按照试剂盒说明书从胚胎肾脏中分离总RNA。使用生物分析仪2100（Agilent Technologies，美国）对总RNA进行质控分析；下一代RNA测序（Next generation RNA sequencing, NGS RNA-seq）由Genewiz Biotechnology（中国苏州）完成。测序在Illumina Hiseq平台上开展，采用2×150 bp双端测序模式，单样本测序数据量约为2000万reads。

按照试剂盒说明书，采用TRIzol试剂（Invitrogen）提取每个样本的总RNA。

下一代测序文库的构建严格按照试剂盒说明书进行。Poly(A) mRNA的富集采用Poly(A) mRNA Magnetic Isolation Module或rRNA Removal Kit完成。mRNA的片段化与反转录引物退火使用First Strand Synthesis Reaction Buffer及Random Primers进行。使用ProtoScript II Reverse Transcriptase合成第一链cDNA，随后采用Second Strand Synthesis Enzyme Mix合成第二链cDNA。经磁珠纯化的双链cDNA使用End Prep Enzyme Mix进行单步反应，完成末端修复及dA尾加尾；随后通过T-A连接反应在DNA两端添加测序接头。对连接有接头的DNA进行磁珠分选，回收长度约420 bp的片段（插入片段约300 bp）。随后使用P5和P7引物对每个样本进行13个循环的PCR扩增：两类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引物携带6碱基索引序列，可实现多样本多重测序。PCR产物经磁珠纯化后，使用Qsep100（Bioptic，中国台湾）进行文库质量验证，并通过Qubit 3.0 Fluorometer（Invitrogen，美国加利福尼亚州卡尔斯巴德）进行文库浓度定量。

为去除接头、PCR引物及其片段等技术序列，并过滤掉碱基质量值低于20的低质量序列，使用Cutadapt（V1.9.1）对fastq格式的原始测序数据进行质控处理，得到高质量清洁数据。

首先，从UCSC、NCBI、ENSEMBL等基因组数据库下载相关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（v2.0.1）构建参考基因组索引。最后，使用Hisat2（v2.0.1）将清洁数据比对至参考基因组。

首先从已有gff注释文件转换得到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，使用HTSeq（v0.6.1）基于双端清洁数据估算基因及转录本异构体的表达水平。