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Sample GSM4419696

Query DataSets for GSM4419696

Status

Public on Oct 06, 2020

Title

Osteoclast-Day1-bulkRNAseq-1

Sample type

SRA

Source name

osteoclasts

Organism

Mus musculus

Characteristics

strain: C57BL/6
tissue: In vitro cultured osteoclasts (24h after RANKL stimulation)
age: 8 weeks old

Extracted molecule

total RNA

Extraction protocol

This osteoclast culture was performed on collagen-coated dishes, which permitted a harvesting of the adherent cells by enzymatic treatment and then a preparation of the single-cell suspensions. Murine bone marrow cells were cultured with M-CSF for 2 days, and these cells were further cultured for 3 days with RANKL in the presence of M-CSF. scRNA-seq, bulk RNA-seq and proteome analyses were performed at the following time points: Day 0 (before RANKL stimulation), Day 1 (1 day after RANKL stimulation) and Day 3 (3 days after RANKL stimulation). Bulk-RNA seq analysis was also performed using control and Cited2-KO cells on Day 3 of the osteoclastogenic culture system to investigate the role and point of action of Cited2 in the trajectory of osteoclastogenesis.
RNA libraries were prepared for sequencing using standard Ion Torrent Proton protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent Proton

Data processing

Single-cell RNA-seq was performed on cells on Day 0, Day 1 and Day3 of the osteoclast culture system with a target output of 3,000 cells/ per time point. Single cells were captured with the 10x Genomics Chromium system. The sequencing library was generated using the 10x Genomics Single Cell 3′ Solution (version 2) kit and subjected to Illumina sequencing (HiSeq 4000). Alignment, quantitation and aggregation of sample count matrices were performed using the 10x Genomics Cell Ranger pipeline as the manufacturer’s protocol (GENEWIZ JAPAN Corp.). For bulk-RNA-seq, cDNA and sequencing libraries were processed using FastQC (v0.1.1.8), TrimGalore (v0.6.4) and Kallisto (v0.46.0) with the mouse transcriptome index (mus musculus GRCm38.96), and analyzed by DESeq2 (v1.26) to quantify gene expression with PCA, hierarchical clustering and differential expression analysis (using log fold change shrinkage and adjusted p-value <0.05).
Supplementary_files_format_and_content: Excel files include expression values for each sample

Submission date

Mar 18, 2020

Last update date

Oct 06, 2020

Contact name

Masayuki Tsukasaki

Organization name

The University of Tokyo

Department

Department of Immunology Graduate School of Medicine and Faculty of Medicine

Street address

7-3-1 Hongo Bukyo-ku