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Sample GSM5057880

Query DataSets for GSM5057880

Status

Public on Feb 11, 2021

Title

HT-29_CoCl2_mRNA_rep3

Sample type

SRA

Source name

HT-29 cells

Organism

Homo sapiens

Characteristics

treatment: CoCl2 for 24 hours
cell line: HT-29

Treatment protocol

A fresh stock solution 0.3 M cobalt chloride (CoCl2) was prepared in water and added to the medium to obtain desired final concentration 300 µM for 24 hours. The second treatment included a fresh portion containing oxyquinoline derivative 4896-3212 (ChemDiv Research Institute, Khimki, Russia) in DMSO (10 mM). The final concentration of oxyquinoline in the medium was 5 μM (0.5 μl of the solution in DMSO per 1 ml medium). In the control, the cells were incubated in a medium with 0.5 μl DMSO per 1 ml medium (without CoCl2 or oxyquinoline).

Growth protocol

HT-29 cells (ATCC, Manassas, VA, USA) were grown in McCoy's 5A medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% non-essential amino acids, penicillin (100 U/mL) and streptomycin (100 mg/mL). Caco-2 cells were obtained from the Russian Vertebrate Cell Culture Collection (Institute of Cytology, Russian Academy of Sciences, Russia). The cells were incubated under conditions for differentiation for 21 days in Eagle’s MEM with 20% FBS, 2 mM glutamine, 0.1 mM non-essential amino acids, 0.1% penicillin-streptomycin. All cell culture reagents were obtained from Gibco, Waltham, MA, USA. Both cell lines were maintained in a humidified atmosphere at 37ºC and 5% CO2, changing medium every 3 days.

Extracted molecule

total RNA

Extraction protocol

Cells were lysed with the Qiazol Lysis Reagent (Qiagen, Hilden, Germany) for subsequent extraction of RNA using the Qiagen miRNeasy Mini Kit (Qiagen, Hilden, Germany). Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) was used to assess quality (260/280) and quantity. Total RNA samples were also QC checked using the Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA).
Libraries for mRNA sequencing were prepared from total RNA samples using Illumina Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). Each sample was sequenced on the Illumina NextSeq 550 to generate single-end 75 nucleotide reads. Libraries for miRNA sequencing were prepared from total RNA samples using NEBNext Multiplex Small RNA Library Prep Kit for Illumina. Each sample was sequenced on the Illumina NextSeq 550 to generate single-end 50 nucleotide reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Description

mRNA_counts.tsv
mRNA_RPKM.tsv

Data processing

Quality of FASTQ files were assessed with FastQC v0.11.9, three miRNA-seq replicates (Caco-2_Control_miRNA_rep3, HT-29_CoCl2_miRNA_rep1 and HT-29_Oxy_miRNA_rep3) had not passed the quality control
Adapters were trimmed with cutadapt v2.10
Trimmed mRNA-seq reads were mapped on the reference human genome (GENCODE GRCh38.p13) with STAR v2.7.5b, GENCODE genome annotation (release 34) was used to generate the count matrix.
MiRNA count matrix was generated by miRDeep2 v2.0.1.2 with the use of bowtie v1.1.1 and miRBase v22.1
Sequencing library sizes were normalized with the Trimmed Mean of M-values (TMM) algorithm available in edgeR v3.30.3 package. The same package was used to generate TMM-normalized Reads Per Kilobase of transcript per Million mapped reads (RPKM) and Counts Per Million mapped reads (CPM) matrices for mRNA-seq and miRNA-seq data, respectively.
Genome_build: GRCh38.p13
Supplementary_files_format_and_content: Tab-separated matrices containing raw read counts and TMM-normalized (edgeR) RPKM and RPM values

Submission date

Feb 01, 2021

Last update date

Feb 12, 2021

Contact name

Stepan Nersisyan

E-mail(s)

[email protected]

Organization name

HSE University

Department

Faculty of Biology and Biotechnology