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Sample GSM3769184

Query DataSets for GSM3769184

Status

Public on Nov 08, 2022

Title

NB4_24h_dmso_5

Sample type

SRA

Source name

NB4 APL cell line

Organism

Homo sapiens

Characteristics

cell line: NB4
cell type: acute promyelocytic leukaemia (APL)
treatment: DMSO
time: 24h
replicate: 3

Treatment protocol

Cells were treated with 1µM of all-trans retinoic acid (ATRA) or DMSO control for 5 days in biological triplicate. Cultures were supplemented with media containing fresh drug on Day 3.

Growth protocol

NB4, NB4-LR2 and NB4-MR2 cells were cultured in RPMI 1640 (Nacalai Tesque) supplemented with 10% FBS (Biowest), 100U/mL penicillin-streptomycin and 2mM L-glutamine (Gibco).

Extracted molecule

total RNA

Extraction protocol

Cells were harvested at the indicated timepoints and RNA was extracted using RNEasy Mini Kit (Qiagen) with on-column DNAse digestion. The RNA was further concentrated and purified with RNEasy Micro kit (Qiagen).
RNAseq libraries were prepared from 33 NB4 APL cell line samples using the Illumina Tru-Seq Stranded Total RNA kit protocol, according to the manufacturer’s instructions (Illumina, San Diego, California, USA). Library fragment size was determined using the DNA 1000 Kit on the Agilent Bioanalyzer (Agilent Technologies). Libraries were quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems). Libraries were pooled in equimolar and cluster generation was performed on the Illumina cBOT system (Illumina). Sequencing (150bp pair-end) was performed on the Illumina HiSeq 3000 system at the Duke-NUS Genome Biology Facility (Singapore), according to manufacturer’s protocol (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Data processing

Sequence reads were trimmed for adapter sequence using using trimmomatic software version 0.36, with parameters MAXINFO:35:0.5 MINLEN:35. After trimming, the lanes (technical replicates) were concatenated.
The trimmed reads were mapped to the Genome Reference Consortium GRCh38 (hg38) using star aligner v2.5.2b, with the chimeric option. The ensembl-90 fasta sequence release was used for indexing, using parameter --runMode genomeGenerate. The fasta files for all chromosomes were downloaded from ftp://ftp.ensembl.org/pub/release-90/fasta/homo_sapiens/dna/ and were concatenated. The parameters for the chimeric star run were: --outFilterType BySJout --outFilterMultimapNmax 20 --twopassMode Basic --alignSJoverhangMin 10 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJstitchMismatchNmax 5 -1 5 5 --chimSegmentMin 12 --chimJunctionOverhangMin 12 --chimSegmentReadGapMax parameter 3 --limitBAMsortRAM 31532137230 --outSAMtype BAM SortedByCoordinate --quantMode GeneCounts
To quantify the aligned reads of the genes, exons and promoters, Feature counts module from the Subread suite v1.5.1 was called, with parameters -t exon -g gene_id -O -s 2 -J -T 8 -p -R -G c GRCh38_r90.all.fa -a ensemble_hg38.gtf. The output file is the RNAseq_full_all.counts.
Genome_build: hg38
Supplementary_files_format_and_content: Raw counts were the output of Feature counts module above. They are provided in the txt file RNAseq_full_all.counts.

Submission date

May 16, 2019

Last update date

Nov 08, 2022

Contact name

Eleni G. Christodoulou

E-mail(s)

eleni.christodoulou@duke-nus.edu.sg, elenichri@gmail.com

Phone

+65 87420979

Organization name

Duke-NUS Medical School