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Identification of quiescent and spatially-restricted mammary stem cells that are hormone responsive

ABSTRACT: Purpose: The aim of this study is to explore histone modification in four basal cell populations defined by Lgr5 and Tspan8 expression taken from adult mouse mammary glands using chromatin immunoprecipitation sequencing (ChIP-seq). Methods: The ChIP DNA samples were prepared and indexed for Ilumina sequencing using the TruSeq DNA sample Prep Kit (Ilumina) as per manufacturer's instruction. The library was quantified using the Agilent Tapestation and the Qubit's RNA assay kit for Qubit 2.0 Fluorometer (Life Technologies). The indexed libraries were then prepared for paired-end 75bp sequencing on a NextSeq500 instrument using the 150 cycle kit v2 chemistry (Ilumina) as per manufacturer's instructions. Reads were aligned to the mouse genome (mm10) using the subread algorithm by Liao et al. (2013) and counted into bins associated with Entrez gene identifiers (27080 genes in total) using featureCounts in two different ways. Genebody counts summarised the number of reads overlapping anywhere between the first and last base of a given gene while promoter counts summarized the number of reads overlapping a region 3000 bases upstream to 2000 bases downstream of the TSS of each gene. For downstream analysis, genebody counts were used for the H3K27me3 marks and promoter counts were used for the H3K4me3 marks.

ORGANISM(S): Mus musculus

PROVIDER: GSE89450 | GEO | 2017/02/01

SECONDARY ACCESSION(S): PRJNA354314

REPOSITORIES: GEO

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Project description:Two tree-man syndrome lesions immersed in RNA later were snap-frozen in liquid nitrogen and crushed with a tissue pulverizer kit (Cell crusher) according to the manufacturer’s instructions. Total RNA was extracted with the RNeasy Fibrous Tissue Mini Kit (QIAGEN), according to the manufacturer’s instructions. The procedure included a DNAse digestion step or an additional column to remove contaminating DNA. The concentration and purity of the total RNA extracted were measured by spectrometry with an Xpose (Trinean). RNA integrity was evaluated by capillary electrophoresis with a Tape Station (Agilent). The Ovation Universal RNA-Seq System from NUGEN was used to prepare the RNA-seq libraries from100 ng of total RNA, as recommended by the manufacturer. This type of RNAseq kit is less sensitive to total RNA degradation (the RNA integrity number (RIN) of the two samples was quite low: 3.9 for one sample and 5 for the other). This kit constructs strand-specific RNA-seq libraries from 10 to 100 ng of total RNA and uses Insert-Dependent Adaptor Cleavage(InDA-C) technology to remove the ribosomal RNA transcripts. The RNA is subjected to reverse transcription and the second strand is synthesized and a fragmentation step is then performed before Illumina-compatible indexed adaptator ligation. This ligation is followed by a strand-selection enzymatic reaction to provide information about the sense of the transcripts. Insert-dependent adaptor cleavage (InDA-C)-specific primers were then used for the targeted depletion of human ribosomal RNA sequencing transcripts before PCR enrichment. We ensure that no excess amplification occurred during the final PCR step, by evaluating the number of PCR cycles to be applied to each sample in a preliminary Q-PCR test with EvaGreen. An equimolar pool of the final indexed RNA-Seq libraries was sequenced on an Illumina NovaSeq6000 (paired-end reads, 100 bases + 100 bases) and ~50 million paired-end reads per library were produced.

Project description:Background. Steer progeny suckled by cows fed a dried distillers grains and solubles (DDGS) diet the first three months of lactation were heavier during feedlot finishing and had significantly lower marbling and larger longissumus muscles than steer suckled by cows fed a control diet (CON). These differences were profound in that progeny were managed and fed identically from weaning until finishing, and suggested that the developmental program of muscle composition was established during the suckling period. Purpose. Transcriptomes of longissimus muscle were measured using next generation sequencing to investigate whether there were any developmental clues to the differences in marbling scores and muscle content between steers suckled by DDGS (n=5) versus control (CON; n=5) diet fed cows during lactation. Methods. Multiparous Angus × Simmental cows with male progeny were fed one of two diets supplemented with either DDGS or soybean meal (CON), from calving until day 129 postpartum (PP). After conclusion of the treatments at 129 days postpartum, cow–calf pairs were comingled and managed as one group until weaning at 219 days postpartum. Steers were then transitioned to a common diet. Longissimus muscle (LM) biopsies were obtained from steers suckled by cows fed DDGS (n=5) and CON (n=5) 10 d prior to slaughter. RNA was extracted from LM. Total RNA isolated and Library Prep Kit was used to generate library for measuring messenger RNA molecules. Library was sequenced on an Illumina HiSeq2500 platform. FastQC (v 0.10.1) was used to assess sequence quality, and quality trimming was done using FASTX toolkit (v 0.0.13). Bases with less than Phred33 score of 30 were removed and resulted in reads of at least 50 bases. Reads were mapped against the bowtie2-indexed Bos taurus genome using Tophat (v 2.0.9) with default parameters, and the genome annotation file (.gtf) from Ensembl was used to associate the genome with annotated features. Raw read counts of each sample for each gene feature were generated with HTSeq (v 0.5.3p7) using Tophat output and Bos taurus annotations. Counts were merged using custom perl scripts and used for downstream differential gene expression (DE) analysis. DE pairwise analysis between samples CON and DDGS was done with ‘R’ (Version 2.15.2). Counts matrix, library sizes, and experimental design were combined to create an object using edgeR (v 3.0.3) package. Results. Number of reads per sample ranged from 79,530,352 to 58,600,824. After trimming there were 77,569,706 to 57,307,614 per sample. The percent of reads that mapped to bovine genome per sample ranged from 77.3 to 92.3%, and averaged 86.3% across all samples. Number of reads per gene ranged from one to over two-hundred ninety thousand counts per million (CPM) reads. There were 809 genes differentially expressed (P-adj<0.1) between CON and DDGS muscle. Of these 637 were upregulated and 172 downregulated in DDGS relative to CON. Overall the DDGS versus CON muscle transcriptomic signature was pro-myogenic and anti-adipogenic. In particular, myokines/satellite cell maintenance factors were found among upregulated (LIF, CNTF, FGFB1, EPHB1) genes. The anti-adipogenic signature was typified by the upregulation of anti-inflammatory cytokines and receptors (IL1RAP, IL1RL2, IL13RA2, IL1F10,), and downregulation of expression of inflammation/inflammatory cytokines and receptor (TNF, IL6R CXCL9), which suggests a selection of differentiation pathways away from adipogenic line. While the upregulation of TGFB, SPP1 and INHBA supports selection of fibroblast lineage of cells. Conclusion. The lactation phase of production can effect meat quality by affecting transcriptional signatures that favor myogenesis and depress inflammation.

Project description:Purpose: The goal of this study is to compare the NGS-derived from ventricular trascriptome profiling (RNA-seq) of 1- and 2-day old mouse hearts to gain insight into the molecular bases responsible for the decline in the heart regeneration competence between P1 and P2 mice Methods: Ventricular cardiac trascriptome profiling were generated by total RNA was extracted from only the ventricles of P1 (n=3) or P2 (n=3) hearts using Quiazol lysis reagent (Quiagen) and RNAeasy extraction kit (Qiagen) according to manufacturer’s instructions. Polyadenylated RNA was isolated from 1-5 μg of total RNA using Dynabeads mRNA purification kit (Invitrogen). Polyadenylated RNA was then fragmented, retro transcribed and finally re-synthetized to double strand DNA molecules using DNA polymerase I (Invitrogen). End-pair, A-tailing, adapter ligation and size selection were then performed. RNA-seq analysis was performed using the 50 nt long paired-end reads obtained from sequencing 6 samples of mice heart tissue at two different time points and using three biological replicates for each time point. Firstly, the quality of the reads was verified using FastQC v0.11.21. Then, the reads were mapped to Mus musculus reference genome vGRCm38 2, using tophat v2.0.143. Following mapping, the transcript and normalization counts were performed using cuffdiff v2.2.1 4. The differential expression analysis of normalized counts was performed using cummeRbund v2.12 5, a widely used R v3.2 package which works together with the cuffdiff software; the applied threshold on the parameter alpha in the function getSig of the package was <= 0.05. Results: We found that most differentially-expressed transcripts encode extracellular matrix components and structural constituents of the cytoskeleton. Conclusion: These results suggest that the stiffness of the local microenvironment, rather than cardiac cell-autonomous mechanisms, crucially determines the ability or inability of the heart to regenerate.

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Identification of quiescent and spatially-restricted mammary stem cells that are hormone responsive

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