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Sample GSM7549616

Query DataSets for GSM7549616

Status

Public on Apr 18, 2024

Title

PB_classical monocytes,SF3B1K700E LR-MDS, patient 3

Sample type

SRA

Source name

PBMCs

Organism

Homo sapiens

Characteristics

disease: LR-MDS
tissue: PBMCs
cell type: classical monocytes (CD14+ CD16-)
gender: male
genotype: SF3B1K700E
treatment: none

Treatment protocol

none

Growth protocol

Monocytes (195000 – 500000 cells per 96-well in 250 µl RPMI 1640/PenStrep/5% heat-inactivated human serum) were cultivated for 4 hours in humidified atmosphere (5% CO2, 37°C).

Extracted molecule

total RNA

Extraction protocol

RNA was isolated with TRIzol and Direct-zol™ RNA Microprep
Ultra-low input RNA sequencing library was prepared by using SMART-Seq HT kit for full-length cDNA synthesis and amplification (Takara, San Jose, CA, USA), and Illumina Nextera XT (Illumina, San Diego, CA, USA) was used for sequencing library preparation. Briefly, cDNA was fragmented, and adaptor was added using Transposase, followed by limited-cycle PCR to enrich and add index to the cDNA fragments. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).
RNA-seq, 2x150 bp paired-end configuration

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Image analysis and base calling were conducted by the HiSeq Control Software. Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into FASTQ files and de-multiplexed using Illumina’s bcl2fastq Conversion Software v2.20. Sequencing quality of raw FASTQ files was checked using FastQC 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapter trimming and quality filtering of sequence reads were performed using Seqtk and cutadapt. The adapter clipped reads were also aligned to ribosomal RNA sequences using Bowtie 2 (v2.4.1) and the mapped reads were discarded. After that, mapping against the human genome (GRCh38 version 32 [Ensembl 98]) was performed using STAR aligner v.2.7.9a.
The R package EBSeq was used for normalization and to infer differential gene and isoform expressions.
Assembly: GRCh38 version 32/Ensembl 98
Supplementary files format and content: Table containing normalized counts for biological replicates

Submission date

Jul 05, 2023

Last update date

Apr 18, 2024

Contact name

Susann Winter

Organization name

University Hospital Carl Gustav Carus, Faculty of Medicine Carl Gustav Carus, TU Dresden

Street address

Fetscherstrasse 74

City

Dresden