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Status |
Public on Sep 11, 2023 |
Title |
RNA-Seq K562 WHITE rep1 |
Sample type |
SRA |
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Source name |
blood
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Organism |
Homo sapiens |
Characteristics |
tissue: blood cell line: K562 disease state: erythroleukemia treatment: untreated agent: untreated
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Treatment protocol |
K562 cells incubated in the presence of 50 μM hemin (neoFroxx, Germany) for 108 h. At this concentration, the drug did not affect cell proliferation.
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Growth protocol |
The cells were drown in RPMI 1640 media (PanEco, Russia) supplemented with heat-inactivated fetal calf serum (HyClone, USA), 2 mM glutamine, 250 u/ml penicillin and 250 μg/ml streptomycin (PanEco, Russia) at 37 °C in a humidified atmosphere containing 5% CO2. For a treated samples, hemin (50 μM, neoFroxx, Germany) was added to induce the erythroid differentiation and cells were incubated further for 108 h. At this concentration, the drug did not affect cell proliferation.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted from cell lysed with Trisol reagent and PureLink RNA Micro Kit (Invitrogen) according to manufacture instruction. RNA Quality was checked with BioAnalyser and RNA 6000 Nano Kit(Agilent). Poly(A)+ RNA was isolated with Dynabeads® mRNA Purification Kit (Ambion). Poly(A)+ RNA was isolated with Dynabeads® mRNA Purification Kit (Ambion). Illumina library was prepared from poly(A)+RNA with NEBNext® Ultra™II RNA Library Prep Kit for Illumina® (NEB) according to manual. Sequencing was performed on Illumina HiSeq1500 with 50 bp read length.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 1500 |
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Description |
Replicate 1 untreated RNAseq.WHITE.forward.bw RNAseq.WHITE.reverse.bw K562.WHITE.TPM.txt K562.WHITE.FPKM.txt
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Data processing |
Base calling and quality control were performed in real time with standard Illumina analysis pipeline using a phiX control. Sequenced reads were trimmed from low quality and/or short sequences and/or Illumina 3' adapters by trimmomatic v. 0.39 using the following options: LEADING:18 TRAILING:18 SLIDINGWINDOW:4:22 MINLEN:20 ILLUMINACLIP:TruSeq3-SE:2:30:10 Trimmed reads were quantified to H. sapiens hg38 GRCh38 Ensembl v.106 genome with Ensembl genome map v.106 by RSEM 1.3.1 using the following options: --fragment-length-mean 255 --star --calc-ci --ci-memory 30720. This operation was performed for both replicates separately. The results were averaged by an in-house R script. Trimmed reads were mapped to H.sapiens genome (hg38/GRCh38 Ensembl v.106) by STAR aligner 2.7.5c with hg38 Ensembl genome map v.106 http://ftp.ensembl.org/pub/release-106/gtf/homo_sapiens/Homo_sapiens.GRCh38.106.gtf.gz. Alignment options were the following: --outSAMunmapped Within --outFilterType BySJout --outSAMattributes NH HI AS NM MD --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --sjdbScore 1 --quantMode TranscriptomeSAM Alignment files were separated by samtools 1.15 view -F 0x10 to get forward strand alignments and by samtools 1.15 view -f 0x10 to get reverse strand alignments. Then bamCoverage (deepTools 3.5.1) tool was applied to obtain normalized expression profiles: --binSize 10 --effectiveGenomeSize 2913022398 --skipNAs --normalizeUsing RPKM --exactScaling. The obtained profiles were averaged between replicates by WiggleTools v1.2.11 and converted to bigWig format by bedGraphToBigWig tool from UCSC genome tools. Assembly: Homo sapiens hg38/GRCh38 Ensembl release 106 genome build. Homo sapiens hg38/GRCh38 Ensembl annotation v106 in GTF format. Supplementary files format and content: bigWig files contain normalized (RPKM, reads per kilobase million) expression signal genomewide for each strand. Supplementary files format and content: Results files contain quantified expression values in TPM (Transcripts Per Million) and FPKM (Fragments Per Kilobase Million) for each gene per replicate and averaged between replicates.
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Submission date |
May 12, 2023 |
Last update date |
Sep 11, 2023 |
Contact name |
Nickolai Tchurikov |
E-mail(s) |
tchurikov@eimb.ru
|
Organization name |
Engelhardt Institute of Molecular Biology
|
Department |
Department of Epigenetic Mechanisms of Gene Expression Regulation
|
Street address |
32, Vavilova str.
|
City |
Moscow |
ZIP/Postal code |
119991 |
Country |
Russia |
|
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Platform ID |
GPL18460 |
Series (2) |
GSE232390 |
Analysis of gene expression changes in K562 erythroleukemia cells after erythroid differentiation caused by incubation in the presence hemin [RNA-Seq] |
GSE232393 |
K562 erythroleukemia cells after erythroid differentiation caused by incubation in the presence of hemin |
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Relations |
BioSample |
SAMN35057780 |
SRA |
SRX20307687 |