C2_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（glucocorticoid steroid hormones）对肺、心脏等众多胎儿器官的成熟与生长发挥关键作用，但目前对其在肾脏中的特异性功能尚缺乏充分表征。糖皮质激素可激活细胞内糖皮质激素受体（glucocorticoid receptor, GR），后者主要作为核转录调控因子发挥功能。本研究通过RNA测序（RNA-seq）分析了胚胎发育第18.5天（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（GR-null, 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）发生显著变化，其中288个基因的绝对对数倍变化（logarithm of fold change, LogFC）>1且FDR < 0.05；进一步分析发现，其中包含16个上调和25个下调的初级纤毛（primary cilia）相关基因（FDR < 0.05）。初级纤毛是一类从细胞膜向外突出的细胞信号与环境感知细胞器，在胚胎发生和组织稳态维持过程中发挥重要作用。目前对调控纤毛发生的细胞通路所知甚少。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发挥调控作用。

我们按照试剂盒说明书，使用TRIzol™试剂(Invitrogen, USA)从胚胎肾脏中分离总RNA。使用Agilent 2100生物分析仪(Agilent Technologies, USA)对总RNA进行质量检测，随后由中国苏州金唯智生物科技(Genewiz Biotechnology)完成下一代RNA测序(next generation RNA-seq, NGS RNA-seq)。本次测序在Illumina Hiseq平台上完成，采用2×150 bp双端测序模式，测序数据量约为2000万条reads。

按照试剂盒说明书，使用TRIzol试剂(Invitrogen)提取每个样本的总RNA。按照制造商提供的实验流程构建下一代测序文库。采用Poly(A) mRNA磁性分离模块(Poly(A) mRNA Magnetic Isolation Module)或核糖体RNA去除试剂盒(rRNA removal Kit)完成poly(A)+ mRNA的分离。使用第一链合成反应缓冲液和随机引物完成mRNA的片段化与引物退火。使用ProtoScript II逆转录酶(ProtoScript II Reverse Transcriptase)合成第一链cDNA，随后使用第二链合成酶混合物(Second Strand Synthesis Enzyme Mix)合成第二链cDNA。经磁珠纯化的双链cDNA随后使用末端修复酶混合物(End Prep Enzyme Mix)进行单管反应，完成两端修复并加上dA尾；之后通过T-A连接反应在两端添加测序接头。随后使用磁珠对连接有接头的DNA进行片段大小筛选，回收约420 bp的片段（插入片段大小约为300 bp）。使用P5和P7引物对每个样本进行13轮PCR扩增；两种引物均带有可与流动槽互补以进行桥式PCR的序列，其中P7引物带有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分析仪(Bioptic, Taiwan, China)进行文库质量验证，并通过Qubit 3.0荧光计(Invitrogen, Carlsbad, CA, USA)进行文库定量。

为去除测序接头、PCR引物及其片段等技术序列，并过滤掉碱基质量值低于20的序列，我们使用Cutadapt(V1.9.1)对fastq格式的原始测序数据进行处理，得到高质量的clean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)对参考基因组序列构建索引。最后，使用Hisat2(v2.0.1)将clean数据比对到参考基因组。

首先，从已有的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)对双端clean数据进行基因及转录本异构体的表达量定量分析。