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Sample GSM3769174 Query DataSets for GSM3769174
Status Public on Nov 08, 2022
Title NB4_4h_atra_4
Sample type SRA
 
Source name NB4 APL cell line
Organism Homo sapiens
Characteristics cell line: NB4
cell type: acute promyelocytic leukaemia (APL)
treatment: ATRA
time: 4h
replicate: 2
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
Street address 8 College Road
City Sinagpore
ZIP/Postal code 169857
Country Singapore
 
Platform ID GPL21290
Series (1)
GSE131325 Identification of drugs in leukaemia differentiation therapy by network pharmacology
Relations
BioSample SAMN11658726
SRA SRX5846220

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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