ena-STUDY-AARHUS UNIVERSITY-26-09-2016-14:09:21:442-453
RNA-seq of Mammary gland cells
The RNA-sequencing was conducted as part of a larger project. A total of 24 samples from four farms divided into three homozygote 3'UTR CSN3 haplotypes were utilized in the RNA-seq analysis. Antibodies was used for precipitation of mammary gland epithelial cells (MEC) whereas centrifugation was used in relation to somatic cells (SC). The precipitation was performed on fresh milk samples. RNA from the cells were used for transcriptome sequencing performed on a HiSeq 2000 (Illumina, San Diego, CA). Trimmed RNA-seq reads were successfully aligned to the cow genome UMD3.1 (Ensembl-82) using STAR and count data was generated. Expectedly, the four caseins were among the most abundantly expressed. In our analysis we defined genes with read counts above 50 across the 24 samples as expressed. A high correlation coefficient was identified between the MEC and SC read counts, hence we decide to use the MEC precipitation for our further analyses. A total of 13,436 genes were expressed in the MEC cells. Further, we also conducted an enrichment analysis of the top 25 genes in order to validate that we did in fact precipitate MEC and we looked at the transcription level of the caseins in order to compare with known protein levels and found a high agreement. A differential expression analysis between the three groups only identified one gene as differential expressed. However, the relative expression of kappa-casein, encoded by CSN3, adjusted for total CSN read count, differed among the haplotypes.
The RNA-sequencing was conducted as part of a larger project. A total of 24 samples from four farms divided into three homozygote 3'UTR CSN3 haplotypes were utilized in the RNA-seq analysis. Antibodies was used for precipitation of mammary gland epithelial cells (MEC) whereas centrifugation was used in relation to somatic cells (SC). The precipitation was performed on fresh milk samples. RNA from the cells were used for transcriptome sequencing performed on a HiSeq 2000 (Illumina, San Diego, CA). Trimmed RNA-seq reads were successfully aligned to the cow genome UMD3.1 (Ensembl-82) using STAR and count data was generated. Expectedly, the four caseins were among the most abundantly expressed. In our analysis we defined genes with read counts above 50 across the 24 samples as expressed. A high correlation coefficient was identified between the MEC and SC read counts, hence we decide to use the MEC precipitation for our further analyses. A total of 13,436 genes were expressed in the MEC cells. Further, we also conducted an enrichment analysis of the top 25 genes in order to validate that we did in fact precipitate MEC and we looked at the transcription level of the caseins in order to compare with known protein levels and found a high agreement. A differential expression analysis between the three groups only identified one gene as differential expressed. However, the relative expression of kappa-casein, encoded by CSN3, adjusted for total CSN read count, differed among the haplotypes.