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Status |
Public on Apr 19, 2022 |
Title |
Sample 3: P1.C.M.3 |
Sample type |
SRA |
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Source name |
Primary CLL cells
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Organism |
Homo sapiens |
Characteristics |
patient: P1 disease state: Chronic Lymphocytic Leukemia treatment: NSC replicate: Rep 3
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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.
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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.
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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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
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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 |
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Relations |
BioSample |
SAMN14652389 |
SRA |
SRX8148309 |