|
| Status |
Public on Nov 04, 2023 |
| Title |
T21/GATA1s/DYRK1A+/+/+ Day 4 megakaryocytes, biol rep 4 [S30] |
| Sample type |
SRA |
| |
|
| Source name |
iPSC
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: iPSC
cell line: TMD8 clone 9
cell type: iPSC-derived CD41+CD42b+ megakaryocytes
genotype: DYRK1A untargeted
treatment: Hematopoietic EB differentiation
|
| Treatment protocol |
Day 7 HPCs and day 4 iPSC-derived megakaryocytes were sorted for CD41+235+ and CD41+CD42b+, respectively.
|
| Growth protocol |
iPSC culture and hematopoietic differentiation by embryoid body (EB) formation were performed as previously described (PMID 25621499, 23045704).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from sorted cells using the RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions.
Library preparation were perform according to the manufacturer’s instructions (Truseq Stranded Total RNA with Illumina Ribo-Zero Plus rRNA Depletion, Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
The RNA-seq data in .fastq files were aligned to the Homo sapiens reference genome GRCh38 and transcriptome using theSTAR(Spliced Transcripts Alignment to a Reference) program. STAR was run in the 2-pass mode to align reads to the reference genome and transcriptome, as well as novel splice junction sites detected by multiple experimental samples. Aligned reads in .bam files were loaded into R and mapped to the sense strand of known exons and genes. A gene-level read count matrix based on uniquely mapped and properly paired reads was used for analysis of differential expression after reads of technical replicates were pooled together. The pooled read counts were converted to FPKM (fragment per kilobase per million reads) to represent gene expression levels.
The R/Bioconductor DESeq2 package was used to test differential gene expression between DYRK1A wild-type and knockout samples. The top differentially expressed genes (DEGs) were selected using a log2 fold change of >2.0, a standard false discovery rate (FDR) of 0.25, and a p-value of <0.01. Gene set enrichment analysis (GSEA) was run on the identified DEGs using gene set collections obtained from BioSystems, KEGG, and MSigDb.V7.4. All genes with detectable expression in the RNA-seq samples were used as background to test enrichment of predefined gene sets.
Samples S18 was determined to be an outlier following exploratory data analysis and was not included in statistical analyses, including differential expression analysis.
Assembly: GRCh38
Supplementary files format and content: One tab-delimited text file of read count matrix by genes (rows) and samples (columns)
|
| |
|
| Submission date |
Sep 21, 2023 |
| Last update date |
Nov 04, 2023 |
| Contact name |
Eric Wafula |
| Organization name |
Children's Hospital of Philadelphia
|
| Department |
DBHi
|
| Street address |
2714-2720 South Street
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19146 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE243702 |
Synergistic roles of DYRK1A and GATA1 in trisomy 21 megakaryopoiesis |
|
| Relations |
| BioSample |
SAMN37494164 |
| SRA |
SRX21848592 |
