|
Status |
Public on May 16, 2022 |
Title |
Empty vector_in-vitro_2 [EV_2] |
Sample type |
SRA |
|
|
Source name |
CD34 positive cells
|
Organism |
Homo sapiens |
Characteristics |
tissue/cell type: CD34 positive cells genotype/treatment: empty vector after five days culture in-vitro sort: GFP+
|
Treatment protocol |
Each CD34 biological replicate composed of three different cord blood units
|
Growth protocol |
Samples 1-4: bone marrow samples from ETV6-NCOA2 patients were transplanted into NSGS mice. Samples 5-12: CD34 cord blood cells were retroviraly infected with ETV6-NCOA2 (NGFR) or ETV6-NCOA2 (NGFR)+NOTCH1-L1601PdP (GFP) and transplanted into NSGS mice. Samples 13-18: The cells were cultured for five days in-vitro (SCF, Flt3L, and TPO) , sorted for GFP positive, and sent for bulk RNA-sequencing.
|
Extracted molecule |
total RNA |
Extraction protocol |
CD34 positive cord blood cells were isolated using magnetic beads from fresh cord blood (For each biological replicate, three different cord blood samples were mixed). The cell were retrovirally infected with ETV6-NCOA2 or empty vector and cultured in IMDM supplemented with hSCF, hTPO, hFlt3L (100 ng/ml) for 5 days. The positive cells were sorted (GFP). ETV6-NCOA2 or ETV6-NCOA2+NOTCH1-L1601PdP were sorted (NGFR+/ GFP+NGFR+ respectively) from fresh bone marrow of engrafted NSGS mice. ETV6-NCOA2 patient derived xenografts were sorted with human CD45 from bone marrow of engrafted NSGS mice. We extracted RNA from the samples and performed bulk RNA-sequencing. Inhouse poly A based RNA seq
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
|
|
Description |
s_2_BB CD34 positive cells transduced with empty vector after five days culture in-vitro
|
Data processing |
Reads were trimmed using cutadapt After trimming the reads were mapped to the human genome using STAR v2.4.2a (using options outFilterMismatchNmax 2 and alignEndsType EndToEnd) Reads whose AS (alignment score) was higher in the mouse compared to human were discarded. The reads were aligned to both GRCm38 sequence and GRCh38 sequences. Reads with a higher mapping quality to the mouse genome were excluded from the analysis. Counting proceeded over genes annotated in Ensembl using htseq-count Normalization was performed using DESeq2 Genome_build: GRCh38 Supplementary_files_format_and_content: Excel file includes the normalized counts
|
|
|
Submission date |
Nov 18, 2020 |
Last update date |
May 16, 2022 |
Contact name |
Gilgi Friedlander |
Organization name |
Weizmann Institute of Science
|
Street address |
Hertzel st
|
City |
Rehovot |
ZIP/Postal code |
76100 |
Country |
Israel |
|
|
Platform ID |
GPL16791 |
Series (2) |
GSE161702 |
ETV6-NCOA2 expression profile in T/M mixed-phenotype acute leukemia [ETV6-NCOA2 human] |
GSE162338 |
ETV6-NCOA2 expression in CD34 positive cord blood cells |
|
Relations |
BioSample |
SAMN16826056 |
SRA |
SRX9526894 |