|
| Status |
Public on May 14, 2024 |
| Title |
subject4_beige_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
primary adipose stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary adipose stem cells
subject: 4
time: day15
treatment: beige adipogenic differentiation
|
| Treatment protocol |
For adipogenic differentiation, confluent cells were cultivated for 72h in DMEM/F12/10% fetal bovine serum without growth factors. For white adipose differentiation ASCs were induced by addition of 10 µg/ml insulin, 200 µM indomethacin, 0.5 µM 1-methyl-3 isobutylxanthine and 1 µM dexamethasone. Medium was renewed every 3 days until day 9. Cells were then maintained in DMEM/F12/10% fetal bovine serum and 10 µg/ml insulin until day 15. For beige adipose differentiation, media were supplemented with 1 µM rosiglitazone until day 15. RNA from the total cell population was harvested by direct lysis in the well.
|
| Growth protocol |
Human normal adipose tissue-derived mesenchymal stem cells were isolated from subcutaneous liposuction material. Cells were expanded in DMEM/F12 containing 10% fetal calf serum, and 20 ng/ml basic fibroblast growth factor (FGF2; Sigma-Aldrich F5392). Cells at passage 5-8 were used in three independent differentiations into the adipocyte lineage.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total mRNA was isolated from cells using the Qiagen RNeasy kit and processed for Illumina library preparation and sequencing.
PolyA-selected RNA was sequenced from paired-end libraries using TruSeq Stranded mRNA kit from Illumina.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
23-22611_S142
|
| Data processing |
Reads were filtered and remaining adaptor sequences trimmed with fastp 0.20.1.
For gene-level analysis reads were aligned to the hg38 genome (GENCODE 32 annotation) with hisat2 (v2.1.0), and counted using featureCounts (v2.0.1). Low abundance genes were filtered using filterByExpr from edgeR and then normalized using the trimmed mean of M values method. Beige and white gene expression was compared pairwise for each donor using the robust eBayes method with limma-voom adjustment.
Genome tracks of Bins Per Million (BPM) mapped reads were generated from hisat alignments with deeptools bamCompare at a binsize of 1 bp for each strand separately. Then differentiation replicates were averaged with BigwigAverage.
For exon-exon junction analysis reads were aligned to hg38 genome with STAR v2.7.10b using GENCODE 32 reference annotation (ENCODE options from the STAR manual were used as well as --outSAMstrandField intronMotif --outSAMtype BAM Unsorted). Split reads with at least a 6 bp of anchor sequence and an intron length between 20 bp and 1 Mb were extracted using regtools. Introns sharing splice sites were clustered using LeafCutter’s leafcutter_cluster_regtools.py script, requiring at least 30 reads per library supporting each cluster for introns of up to 1 Mb in size.
Differences in intron excision were tested between white and beige triplicates with donor as a confounding variable. Strict prefiltering required that an intron was found in at least 14 (of 36) libraries and that clusters had at least 12 libraries per condition with 20 or more spliced reads. The differential_splicing function from LeafCutter R package v0.2.9 was used to implement the Dirichlet-multinomial generalized linear model (15). LeafCutter assigns a ∆PSI to each intron and a p-value to each cluster, which contains introns that share splice sites and thus belong to alternative isoforms of that gene or genes. Effect size and cluster significance output files were merged to make ∆PSI and cluster p-value information available in one table. Introns were assigned to transcripts, with the priority for annotations: GENCODE 32 > RefSeq GCF_000001405.40-RS_2023_10 > FANTOM CAT robust (Hon et al. 2017). In other words, only if an intron was missing from the GENCODE annotation was it compared to the RefSeq annotation and so on.
Normalized TRIFID prediction scores were downloaded for the GENCODE 37 and RefSeq110 annotations. Where multiple transcripts were possible for a single intron excision event, TRIFID scores were averaged, but if no score was found -0.1 was used. To calculate a TRIFID difference, the top two intron-excision events were compared for clusters with at least one significant junction (| ∆PSI | > 0.1 & p.adjust < 0.05).
Assembly: hg38 GENCODE 32
Supplementary files format and content: .fwd.bw Bins Per Million (BPM) mapped reads averaged across three differentiation replicates. Read pairs mapped to the forward strand only. Binsize 1bp.
Supplementary files format and content: .rev.bw Bins Per Million (BPM) mapped reads averaged across three differentiation replicates. Read pairs mapped to the reverse strand only. Binsize 1bp.
Supplementary files format and content: Counts of reads in genes from gencode 32 annotation.
Supplementary files format and content: Fragments per kilobase per million (FPKM) for each gene passing the filtering threshold in each sample.
Supplementary files format and content: perind_numers.junction.counts.gz - Counts of reads spanning each exon-exon junction.
Supplementary files format and content: Information from leafcutter on each exon-exon junction (e.g. cluster id, deltaPSI and adjusted pvalue), and annotation with reference databases and intron numbers.
|
| |
|
| Submission date |
Feb 21, 2024 |
| Last update date |
May 14, 2024 |
| Contact name |
Philippe Collas |
| Organization name |
University of Oslo
|
| Department |
Institute of Basic Medical Sciences
|
| Street address |
PO Box 1112 Blindern
|
| City |
Oslo |
| ZIP/Postal code |
0317 |
| Country |
Norway |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE256259 |
Differential junction usage in paired RNA-seq of white and beige differentiated adipocytes from six human subjects |
| GSE256262 |
Alternative isoform expression of key thermogenic genes in human beige adipocytes |
|
| Relations |
| BioSample |
SAMN40020102 |
| SRA |
SRX23685505 |
