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Sample GSM6918284

Query DataSets for GSM6918284

Status

Public on Jan 18, 2023

Title

AmmoniaMPs_miRNA_biol rep 1 [NH3_MPs_1_S4]

Sample type

SRA

Source name

CHO-K1

Organism

Cricetulus griseus

Characteristics

cell line: CHO-K1
cell type: Chinese Hamster Ovary (CHO) cells
treatment: Added 9 mM Ammonium Chloride at start of culture

Growth protocol

CHO cells were cultured in HyClone™ ActiPro™ Media supplemented with 6 mM L-glutamine at 200 RPM, 37°C, and 5% CO2. Ammonium chloride (NH4Cl) dissolved in PBS (pH 7.4), was added to ammonia stressed cultures at the start of culture for a starting concentration of 9 mM NH4Cl. Cultures were seeded at a density of 0.4 x 106 cells/mL in a total volume of 15 mL. Cultures were harvested on day 3 for samples (cells, MPs, exosomes).

Extracted molecule

total RNA

Extraction protocol

Followed protocol of TruSeq Small RNA Sample Prep Kit (Illumina)
Following isolation of cells, MPs, and exosomes, total RNA was isolated with the miRNeasy micro kit (Qiagen). RNA concentration was determined using the Qubit RNA High Sensitivity (HS) kit and size distribution of total RNA was determined by fragment analysis (AATI Fragment Analyzer, Agilent). The 9 standard culture sample libraries were pooled together. The 18 osmolarity and ammonia stressed sample libraries were pooled together.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

NextSeq 550

Description

counts-20221123 reformat.xlsx
expression-table-export-20221123.xlsx

Data processing

Quality of sequencing data was assessed using FastQC (ver. 0.10.1; Babraham Bioinformatics). All reads were mapped to the Cricetulus griseus genome PICR assembly (RefSeq GCF_003668045.1, Annotation version 103) to determine the overall biotype (e.g. mRNA, miRNA, etc.) composition of reads using CLC Genomics Map Reads to Reference tool with default settings (ver 20.0.2). For samples from standard cultures, candidate smRNA sequences were identified from raw sequence data using cutadapt (ver. 2.8) with parameters “-u -4 -a A{10}” as recommended by PerkinElmer NextFlex Combo-Seq kit. All reads shorter than 50 bp and containing the poly-A adapter were utilized for miRNA analysis. Reads passing filter were analyzed using the CLC Genomics Server (ver. 20.0.2; Qiagen Bioinformatics). Samples were processed in batch using CLC Quantify miRNA with “strand specific=yes” and allowing up to 2 mismatches. For miRNA, miRbase (ver. 22.1) was used as a reference database with species in this priority order: C. griseus, Mus musculus, Rattus norvegicus, and Homo sapiens and results were grouped on mature miRNA exact matches. Differential expression analysis utilized TMM normalization and the CLC Differential Expression Analysis based on negative binomial Generalized Linear Model (GLM) using the Wald test for determining statistical significance and FDR correction.
Assembly: For miRNA, miRbase (ver. 22.1) was used as a reference database with species in this priority order: C. griseus (CriGri_1.0), Mus musculus (GRCm38), Rattus norvegicus (Rnor_6.0), and Homo sapiens (GRCh38) and results were grouped on mature miRNA exact matches.
Supplementary files format and content: counts-20221123 reformat.xlsx contains the total counts and counts per million (CPM) of each microRNA for all samples
Supplementary files format and content: expression-table-export-20221123.xlsx contains differential analysis comparisons between standard and stressed samples

Submission date

Jan 05, 2023

Last update date

Jan 18, 2023

Contact name

Jessica Belliveau

E-mail(s)

jbellive@udel.edu

Organization name

University of Delaware

Department

Dept. of Chemical and Biomolecular Engineering