|
| Status |
Public on Apr 19, 2022 |
| Title |
Sample 9: P1.T.M.3 |
| Sample type |
SRA |
| |
|
| Source name |
Primary CLL cells
|
| Organism |
Homo sapiens |
| Characteristics |
patient: P1
disease state: Chronic Lymphocytic Leukemia
treatment: siZAP70
replicate: Rep 3
|
| Treatment protocol |
For samples with anti-IgM treatment, primary CLL cells were co-cultured with EL08 stromal cells and treat with anti-IgM 4ug/1x10^6 CLL cells.
|
| Growth protocol |
Primary CLL cells were transfected with NSC or ZAP-70 siRNA and co-cultured with EL08 stromal cells in RPMI 1640 medium for totally 7 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Primary CLL cells were harvested after positivley selected by anti-CD19 beads and total RNA was extracted using the standard protocol from GenElute Mammalian Total RNA Miniprep Kit (Sigma/RTN70).
NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB #7420) was used with 100ng of total RNA for the construction of sequencing libraries. mRNA was isolated and purified by using NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB #7490) before cDNA synthesis.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
The quality of the data was assessed using FastQC, MultiQC, PCAs, Jaccard Similarity Indices and MA plots
the reads were aligned using STAR v2.7.3a (parameters --outSAMtype BAM SortedByCoordinate --outReadsUnmapped Fastx --readFilesCommand gunzip -c)
Protein-coding gene quantification was performed using Subread featureCounts v2.0.0 (default parameters)
The raw expression levels were normalized with edgeR v3.26.8. After normalization, a noise filtering step was applied i.e. for each gene, if the maximum normalised expression level was <10 then the gene was excluded from the downstream analysis.
Statistical testing for differential expression of genes was performed using edgeR, with multiple-testing correction based on the Benjamini-Hochberg method. DE Genes were called on adj p-value < 0.05, and |log2(FC)| > 0.5.
The enrichment analysis was performed using g:profiler (Reimand et al., Nucleic Acids Research, 2016)
Genome_build: GRCh38.p13
Supplementary_files_format_and_content: raw.csv contains raw gene counts; normalized.csv contains edgeR-normalized expression levels after the application of the noise filter
|
| |
|
| Submission date |
Apr 21, 2020 |
| Last update date |
Apr 19, 2022 |
| Contact name |
Irina Mohorianu |
| E-mail(s) |
data-submissions@stemcells.cam.ac.uk
|
| Organization name |
University of Cambridge
|
| Department |
Wellcome-MRC Cambridge Stem Cell Institute
|
| Street address |
Puddicombe Way
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0AW |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE149020 |
Next generation sequencing quantitatively analyzes transcriptomes of primary CLL cells tranfected with non-specific control (NSC) or ZAP-70 siRNA (-anti-IgM dataset) |
| GSE149022 |
Next generation sequencing quantitatively analyzes transcriptomes of primary CLL cells tranfected with non-specific control (NSC) or ZAP-70 siRNA |
|
| Relations |
| BioSample |
SAMN14652398 |
| SRA |
SRX8148312 |
