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Sample GSM7441676

Query DataSets for GSM7441676

Status

Public on Jun 07, 2023

Title

HAP1_WT_rep1

Sample type

SRA

Source name

cell line

Organism

Homo sapiens

Characteristics

tissue: cell line
cell line: HAP1 (RRID:CVCL_Y019)
cell type: Myeloid haploid karyotype cell line
genotype: WT

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.

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

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

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

Street address

Lazarettgasse 14

City

Vienna