Total RNA-sequencing in TC28a2 cell line with or without hypertrophic medium

ABSTRACT: We report the results of performing RNA sequencing using RNA extracted from TC28a2 cells grown in the presence of hypertrophic differentiation medium compared to RNA extracted from TC28a2 cells grown in growth medium. For preparation of samples for RNA sequencing, total RNAs were extracted from TC28a2 cells treated with growth medium (n=2) or hypertrophic medium (n=3) at day 5 of differentiation. RNA was extracted using Trizol (Invitrogen) following the protocols provided by the manufacturer. Isolated RNAs were submitted to the Macrogen Inc. (Macrogen Inc., Seoul, South Korea) for total RNA-sequencing. The overall quality of the extracted total RNAs were validated using spectrophotometry. To remove low quality and adapter sequence, the raw was read by the sequencer before analysis and align the processed reads to the Homo sapiens using HISAT2 v2.1.059. The reference genome sequence of Homo sapiens (hg19) and annotation data were downloaded from the NCBI, and transcript assembly of known transcripts was then processed by StringTie v2.1. Base on the result of that, expression abundance of transcript and gene were calculated as read count or FPKM value (Fragments Per Kilobase of exon per Million fragments mapped) per sample. The expression profiles are used to do additional analysis such as DEG (Differentially Expressed Genes). The relative abundances of gene were measured in read count using StringTie. Genes with one more than zeroed read count values in the samples were excluded. Filtered data were log2-transformed and subjected to RLE Normalization. Statistical significance of the differential expression data was determined using DESeq2 nbinomWaldTest and fold change in which the null hypothesis was that no difference exists among groups. False discovery rate (FDR) was controlled by adjusting p value using Benjamini-Hochberg algorithm. For DEG set, hierarchical clustering analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Enrichment of gene ontology analysis was performed for DEGs using g:Profiler and KEGG pathway analysis was tested based on KEGG pathway (https://www.genome.jp/kegg/) database. We used multidimensional scaling (MDS) method to visualize the similarities among samples. We applied to the Euclidean distance as the measure of the dissimilarity. Hierarchical clustering analysis also was performed using complete linkage and Euclidean distance as a measure of similarity to display the expression patterns of differentially expressed transcripts which are satisfied with fold change ≥2 and raw p <0.05. All data analysis and visualization of differentially expressed genes was conducted using R 3.6.1(www.r-project.org).

ORGANISM(S): Homo sapiens

PROVIDER: GSE199847 | GEO | 2022/04/03

REPOSITORIES: GEO