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Sample GSM6436672

Query DataSets for GSM6436672

Status

Public on Oct 23, 2023

Title

1C4MC

Sample type

SRA

Source name

cell line

Organisms

Homo sapiens; Mus musculus

Characteristics

tissue: cell line
cell line: C4-2B;MC3T3-E1
cell type: human prostate cancer cells; mouse osteoblasts
genotype: WT
treatment: Co-culture in 1:30 ratio

Growth protocol

All the cells were maintained in alpha-MEM supplemented with 10% FBS.

Extracted molecule

total RNA

Extraction protocol

Total RNAs were extracted from cells cultured alone or the mixed co-culture using RNeasy kit and were decontaminated of the DNA using RNase-Free DNase Set (Qiagen, Hilden, Germany). 500 ng of total RNA was used for the construction of sequencing libraries.
Libraries were prepared by the Van Andel Institute Genomics Core from 500 ng of total RNA using the KAPA stranded mRNA kit (v4.16) (Kapa Biosystems, Wilmington, MA). RNA was sheared to 300-400 bp. Prior to PCR amplification, cDNA fragments were ligated to IDT for Illumina UDI adapters (IDT, Coralville, IA). Quality and quantity of the libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Santa Clara, CA) and QuantiFluor® dsDNA System (Promega). Individually indexed libraries were pooled and 50 bp, paired end sequencing was performed on an Illumina NovaSeq 6000 sequencer using 100 cycle sequencing kit (Illumina Inc., San Diego, CA) to a minimum read depth of 40 M reads/library.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

We applied a customed species-specific pipeline by integrating three different methods, Xenome, Xenofilter, and Kallisto, to extract human- and mouse- specific reads from the RNA-Seq data from the mixed co-culture samples. For Xenome and Xenofilter, read alignment was performed based on reference genome (GRCh38 for human and GRCm38 for mouse) and human/mouse-only reads were sorted with default settings and the species-specific reads were used for downstream differential expression (DE) analyses. For Kallisto, a combined human-mouse transcriptome was created by joining human transcriptome and mouse transcriptome (downloaded from Gencode) and then the FastQ reads were mapped to the joint transcriptome. Human/mouse-specific transcript counts were extracted for downstream DE analyses. DE analyses were performed with DESeq2 in R and DE gene thresholds were set at |log2FC| >=1 & padj < 0.05.
Assembly: GRCh38/GRCm38
Supplementary files format and content: CSV file containing log2FC results from DESeq2

Submission date

Aug 08, 2022

Last update date

Oct 23, 2023

Contact name

Shang Su

E-mail(s)

shang.su@utoledo.edu

Organization name

The University of Toledo

Department

Cell and Cancer Biology