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|Public on Feb 09, 2018
|Comprehensive mapping of transcriptional networks specifying lactogenic differentiation of murine mammary epithelial stem like cells
|Expression profiling by high throughput sequencing
|Purpose: Understanding of gene transcriptional networks specifying murine mammary epithelial cells (HC11) during lactogenic differentiation and its comparision with murine embryonic stem cells
Methods: RNA-sequencing after ribosomal RNA depletion followed by cDNA synthesis, adaptor ligation and sequencing in duplicate of each sample using Illumina Hi-seq 2500 paired end sequencing. Sequence reads with adapters, over-represented and low-quality reads were discarded. High quality reads were mapped to reference mouse genome (mm10) using Bowtie followed derivation of FPKM values using cufflinks and DE-seq to observe differentially expressed genes.
Results: Using standard analysis methods which were published recently we uniquely mapped more than 50 million sequence reads out of which total 11961 transcripts were from ESC, 11045 from Normal, 11243 from Primed and 10719 from prolactin stage. When we did DE-seq differential gene expression analysis we observed that for Normal to primed transition 1141 and 1026 transcripts were Up and Down regulated respectively where as upon transition from Primed to Prolactin stage 385 and 655 transcripts were Up and Down regulated respectively. We also did pathway analysis to interpret the transcript activation and its function during lactogeinc differentiation; we found that the pathways which are highly up regulated are related to carbohydrate, lipids, nucleic acid and protein metabolism. We also observed that most of the up regulated pathways were related to maintenance of mammary cells differentiated state and nutrition of the infants. Our study concludes that overall differentiation requires priming with glucocorticoids followed by prolactin stimulation of HC11 cells, which tunes the cells for the secretion of milk and its maintenance of the differentiated state till the lactation period.
Conclusions: Transcriptome analysis by next generation sequencing allowed us to explore global gene expression during lactogenic differentiation of HC11 cells in presence of glucocortiocids in primed state. Glucocorticoids are activating genes, transcription factors, epigenetic modifiers and metabolic pathways which are required for later stages of lactation. On other hand gene expression analysis of prolactin stage revealed that there is a focused gene expression exclusively related to lactation process. We also observed that HC11 stem like marker are highly expressed in undifferentiated state whose expression was down regulated in primed and prolactin state indicating that HC11 are partially bipotent cells; when these cells receive differentiation signals they loose their stem cell like characteristics and are differentiated.
|Paired end sequencing of transcriptome in replicates of embyonic stem cells, normal, primed and prolactin. Analysis of data and validation by real-time pcr analysis.
|Sornapudi T, Nayak R, Uppada V, Guthikonda PK, Kethavath S, Yellaboina S, Pasupulati AK, Kurukuti S
|Nov 28, 2017
|Last update date
|Jun 14, 2019
|University of Hyderabad
|Illumina HiSeq 2500 (Mus musculus)