DOI: 10.5061/dryad.v9s4mw74c
title: Gene expression analysis from splenocytes treated with mPVAT-conditioned media

description: The overall purpose of this study was to understand the impact of a
 high-fat diet on the perivascular microenvironment in Dahl S rats, a
 spontaneously hypertensive rat strain. Specifically, the study was
 designed to understand whether a high-fat diet promotes inflammation in
 the perivascular adipose tissue microenvironment. To address this, we
 collected mesenteric PVAT from Dahl S rats on either a control or a
 high-fat diet and used the PVAT to make conditioned media. We then
 cultured rat splenocytes in either mPVAT-conditioned media or in standard
 tissue culture media and activated them with a T cell-specific activator
 (anti-CD3/anti-CD28). We then isolated RNA from the splenocytes which was
 used for gene expression analysis by RNA-sequencing.
description: Primary splenocytes were activated with a T cell-specific
 activator in the presence and absence of perivascular adipose tissue
 (PVAT)-conditioned media. The PVAT was collected from Dahl S rats on
 either a control or high-fat diet for 10 weeks. RNA from the cells was
 extracted using the RNeasy Mini Kit (Qiagen, Germantown, MD).
 Transcriptome analysis of RNA sequencing (30M raw reads/sample) was
 performed via Illumina platforms by Novogene (Sacramento, CA). After
 quality control, HISAT2 was used to map the filtered sequenced reads to
 the reference genome. Gene expression level is estimated by the FPKM
 (Fragments Per Kilobase of transcript sequence per Millions base pairs
 sequenced) method. The coexpression Venn diagram was used to visualize the
 number of genes that are uniquely expressed within each group/sample.
 Differentially expressed genes (DEGs) analysis of HF condition and CTL
 condition was performed using the DESeq2 R package. Volcano plots were
 used to infer the overall distribution of DEGs. Hierarchical clustering
 analysis was carried out of log2(FPKM+1) of union DEGs, within all
 comparison groups.
description: # Gene expression analysis from splenocytes treated with mPVAT-conditioned
 media
 [https://doi.org/10.5061/dryad.v9s4mw74c](https://doi.org/10.5061/dryad.v9s4mw74c) Overview:  The dataset includes RNA-sequencing data from splenocytes treated with perivascular adipose tissue-conditioned media. The PVAT was collected from Dahl S rats on either a control diet or a high fat diet for 10 weeks. Study design: The study is designed to understand the effect of the perivascular adipose tissue (PVAT) on T cell function in the context of high fat diet-induced hypertension. Male Dahl S rats were put on a control diet or high fat diet for 10 weeks after which mesenteric perivascular adipose tissue (mPVAT) was collected to make conditioned media. Splenocytes were cultured in the mPVAT-conditioned media and activated with a T cell-specific activator (anti-CD3/anti-CD28). RNA was isolated from the splenocytes for bulk RNA-sequencing. The data are organized as splenocytes treated with PVAT-CM from rats on control diet vs splenocytes treated with PVAT-CM from rats on high fat diet. ## Description of the data and file structure List of files: 1.     QC_Summary.xlsx 2.     gene.fa 3.     fpkm_sample.xls.xlsx 4.     readcount.xls.xlsx 5.     MapStat_summary.xls.xlsx 6.     HFvsCtrl_DEG_all_PVATCM.xls.xlsx File Descriptions: **1.     QC_Summary.xlsx** The following terms are used in this file: A.    Sample name Male Dahl S rats were placed on control diet or high fat diet for 10 weeks after which mesenteric PVAT was collected and used to make PVAT-conditioned media. Splenocytes were cultured in PVAT-conditioned media from rats on either control diet or high fat diet and activated with a T cell specific activator for 96 h after RNA was extracted and submitted for bulk RNA-sequencing. There are two sample groups: control diet ( C1_4, C2_1, C3_1, C4_1 and C5_1) and high fat diet (HF1_1, HF2_3, HF3_2, HF4_2 and HF5_1). B.    Raw reads The raw reads are just the unfiltered raw reads from sequencing. C.    Clean reads The clean reads are derived from filtering the raw reads. The criteria for filtering were 1. Remove reads with adapters, 2., remove reads containing N &gt; 10% where N = represents a base that could not be determined and 3. Remove low quality reads (where the Qscore of over 50% of the bases is &lt;=5). D.    Raw bases Raw bases are the base number of raw data (number of raw reads * sequence length), converting units to G. E.    Clean bases The clean bases are the base number of raw data after filtering (number of clean reads * sequence length), converting units to G. F.    Error rate Base error rate of whole sequencing. G.   Q20(%) Phred values greater than 20 base number contain the percentage of total bases. (Base number of Phred value &gt; 20)/(Total base number *100) H.    Q30(%) Phred values greater than 30 base number contain the percentage of total bases. (Base number of Phred value &gt; 30)/(Total base number *100) I.       GC content (%) The percentage pg G & C base numbers as a percentage of total bases. (G&C base numer)/(Total base number)*100 **2.     gene.fa** The genes and sequences that were analyzed. **3.     fpkm_sample.xls.xlsx** The fragments per kilobase of transcript per million mapped reads (fpkm) values for each replicate within each group for each gene. **4.     readcount.xls.xlsx** The total read counts for each replicate within each group for each gene. **5.     MapStat_summary.xls.xlsx** Terms used in the MapStat summary: A.    Total reads: total clean reads using for analysis B.    Total mapped reads: numbers of reads being mapped on the genome C.    Uniquely mapped reads: numbers of reads being mapped on single position of the genome D.    Multiple mapped reads: numbers of reads being mapped on multiple positions of the genome E.    Totally mapping rate: (mapped reads)/(total reads)*100, normally the ratio should higher than 70% F.    Uniquely mapping rate: (uniquely mapped reads)/(total reads)*100 G.   Multiple mapping rate: (multiple mapped reads)/(total reads)*100, normally the ratio should lower than 10% **6.     HFvsCtrl_DEG_all_PVATCM.xls.xlsx** Terms used in this table: A.    Gene ID: ID number for each gene B.    Sample groups and replicates. There are two sample groups: control diet (C1_4, C2_1, C3_1, C4_1 and C5_1) and high fat diet (HF1_1, HF2_3, HF3_2, HF4_2 and HF5_1). C.    log2FoldChange: the result performed by DESeq2 R/EdgeR R package D.    pvalue: p-value in statistical tests E.    padj: adjusted p-value. The smaller the p-adjusted is, the more significant differentially expressed genes F.    gene_name: The name of each gene G.   gene_start: The start position of the gene at the chromosome H.    gene_end: The end position of the gene at the chromosome I.       gene_strand: the chain information of the gene J.     gene_length: The length of the gene K.    gene_biotype: gene type L.     gene_description: a description of the function of the gene M.   tf_family: The family name of transcription factor The final columns include the read counts and fpkm for each sample replicate for each gene.