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Status |
Public on Jun 07, 2023 |
Title |
HAP1-ABCB1-KO-c4_rep2 |
Sample type |
SRA |
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Source name |
cell line
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Organism |
Homo sapiens |
Characteristics |
tissue: cell line cell line: HAP1 (RRID:CVCL_Y019) cell type: Myeloid haploid karyotype cell line genotype: ABCB1 KO
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Treatment protocol |
Drug-treated cell lines were grown in the presence of 125 or 500 mM MZ1 (and DMSO as control) for two weeks. During culture, cells were split every 2-3 days and fresh MZ1 was added each time.
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Extracted molecule |
polyA RNA |
Extraction protocol |
At the time of harvest, media is removed from cells and cells are washed once with 1 ml PBS buffer. After washing, 200 μl buffer RLT (Qiagen) is added directly to the cells. After complete cell lysis, lysates are transferred into matrix tubes and frozen at -80 °C until further treatment. For RNA purification, RNeasy 96 Kits (Qiagen) were used. DNA is removed by on-column DNase treatment as described in the manual. After purification, RNA is eluted in 60-80 μl RNase-free water and stored at -80 °C until further usage. RNA samples were normalized on the MicroLab STAR automated liquid platform (Hamilton). Total RNA input of 250 ng was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760, together with the NEBNext Poly(A) mRNA Magnetic Isolation Module #E7490 upstream and the NEBNext Multiplex Oligos for Illumina #E7600 downstream (all New England Biolabs). The only deviation from the manufacturer’s protocol was the use of Ampure XP beads (Beckman Coulter) for double-stranded cDNA purification, instead of the recommended SPRIselect Beads. The index PCR was performed with 12 cycles, while the final library was eluted in 35 μL. mRNA libraries were then quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). mRNA libraries were also assessed for size distribution and adapter dimer presence (<0.5%) by the High Sensitivity Small Fragment DNF-477 Kit on a 96-channel Fragment Analyzer (Agilent). All sequencing libraries were normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina). The library pools were subsequently clustered on S4 Flow Cell and sequenced on a NovaSeq 6000 Sequencing System (Illumina) with dual index, paired-end reads at 2 x 50 bp length (Read parameters: Rd1: 51, Rd2: 8, Rd3: 8, Rd4: 51), aiming for an average depth of 25 million Pass-Filter reads per sample.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
read trimming: cutadapt (version 2.8) read mapping: STAR (version 2.7.9a); reference genome annotation: Ensembl 98 read quantification: RSEM (version 1.3.2) quality control: multiqc (version 1.11), FastQC (version 0.11.9), RSeQC (version 4.0.0) Assembly: GRCh38.p13 Supplementary files format and content: count_table.tsv: tab-delimited text file with read counts per Ensembl Gene ID for each sample Supplementary files format and content: tpm_table.tsv: tab-delimited text file with TPM values per Ensembl Gene ID for each sample
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Submission date |
Jun 02, 2023 |
Last update date |
Jun 07, 2023 |
Contact name |
Giulio Superti-Furga |
Organization name |
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
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Street address |
Lazarettgasse 14
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City |
Vienna |
ZIP/Postal code |
1090 |
Country |
Austria |
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Platform ID |
GPL24676 |
Series (1) |
GSE234043 |
Transcriptional chracterization of ABCC1 and ABCB1 KO and MZ1 treated WT HAP1 cell lines. |
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Relations |
BioSample |
SAMN35573153 |
SRA |
SRX20580094 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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