|
| Status |
Public on Jul 24, 2021 |
| Title |
Sample 12_CD34+ cells Delta1ext-IgG hypoxia bulkRNA rep6 |
| Sample type |
SRA |
| |
|
| Source name |
CD34+ cells Delta1ext-IgG hypoxia bulkRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: G-CSF mobilized human CD34+ cells
treatment: ex vivo cultured for 24 hours with 5 µg/mL Delta1ext-IgG in hypoxia
|
| Growth protocol |
Human CD34+ cells from 6 different healthy donors were thawed and resuspended at a concentration of 5.0 x 105 cells/mL in StemSpanTM Serum-Free Expansion Medium II (STEMCELL Technologies) supplemented with 50 ng/mL of human recombinant Stem Cell Factor (SCF), Fms-like tyrosine kinase 3 ligand (FLT3) and Thrombopoietin (TPO) (PeproTech). Subsequently, cells were ex vivo cultured for 24 hours in vessels coated with Delta1ext-IgG at 10 µg/mL under normoxia and 5 µg/mL under hypoxia.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. The genomic DNA elimination columns contained in the kit were used to eliminate possible DNA contamination during the extraction.
The sequencing libraries were constructed from 10 pg–10 ng of total RNA using the Takara Bio USA’s SMART-Seq V4 Ultra Low Input RNA Kit for Sequencing (Cat # 634888) following the manufacturer’s instructions. Full-length cDNA synthesis was performed using oligo-dT and SMART oligos, and the Illumina sequencing adaptors and barcodes incorporated into the final library by PCR. The fragment size of the RNAseq libraries was verified using the Agilent 2100 Bioanalyzer (Agilent) and the concentrations determined using the Qubit instrument (Thermo Fisher Scientific). The libraries were loaded onto the Illumina HiSeq 3000 machine (Illumina) for paired end 75 bp read sequencing and generated about 50 M reads per sample. The.fastq files were generated using the bcl2fastq software (Illumina) for further analysis.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
DA4562-18_S41
6th biological replicate
|
| Data processing |
Rigorous quality controls of paired-end reads were assessed using FastQC tools [A Quality Control Tool for High Throughput Sequence Data [Online]. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (2015), "FastQC," https://qubeshub.org/resources/fastqc].
Reads were aligned to the reference genome using the latest version of HISAT2 [PMID: 31375807], which is a splice-aware aligner that sequentially aligns reads to the known transcriptome and genome.
FeatureCounts [PMID: 24227677] was used for gene level abundance estimation using the GENCODE (v25) [PMID: 22955987] comprehensive gene annotations.
Principal component analysis (PCA) was used to assess outlier samples. Genes were kept in the analysis if they had raw read counts 5 in at least four samples (for each pairwise comparison performed in the differential expression analysis).
Differential expression analysis at the gene levels of summarization were then carried out using open source Limma R package [DOI: https://doi.org/10.1007/0-387-29362-0_23].
Limma-voom [PMID: 24485249, PMID: 25605792], was employed to implement a gene-wise linear modelling which processes the read counts into log2 counts per million (logCPM) with associated precision weights.
The log2CPM values were normalized between samples using trimmed mean of M-values (TMM) [PMID: 20196867].
We adjusted the differential expression analysis by using a covariate indicating paired samples from subjects following the experimental design.
We adjusted for multiple testing by reporting the FDR q-values for each feature [PMID: 26826716].
Genome_build: Gencode v25
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
|
| |
|
| Submission date |
Sep 03, 2020 |
| Last update date |
Jul 24, 2021 |
| Contact name |
Daisuke Araki |
| E-mail(s) |
[email protected]
|
| Phone |
301-827-1467
|
| Organization name |
National Institutes of Health
|
| Street address |
9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL21290 |
| Series (1) |
| GSE157465 |
bulkRNA seq analysis of adult CD34+ cells ex vivo cultured with Delta1ext-IgG under normoxia or hypoxia |
|
| Relations |
| BioSample |
SAMN16056384 |
| SRA |
SRX9073237 |
