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Sample GSM4396419

Query DataSets for GSM4396419

Status

Public on Mar 09, 2020

Title

SMSC-1d

Sample type

SRA

Source name

skeletal muscle satellite cells

Organism

Columba livia

Characteristics

tissue: pectoral muscles
cell type: skeletal muscle satellite cells
developmental stage: 1d

Treatment protocol

16-day-old pigeon embryos were dissected, followed by removing bilateral pectoral muscles and soaking in PBS (Hyclone, Utah, USA). The pectoral muscles were finely minced and dissociated in Dulbecco’s modified Eagle’s medium(DMEM) containing 0.1% collagenase type IV (Sigma, MO, USA) for about 45 min. Subsequently, cell suspension was filtered twice using a 40-μm nylon mesh (BD, Falcon™) to remove debris. The cells were collected by centrifugation at 1500 r·min–1 for 5 min at room temperature and resuspended in DMEM (Hyclone, Utah, USA) containing 20% fetal bovine serum (FBS) (Natocor, Villa Carlos Paz, Argentina) with 100 U/mL penicillin and 100 μg/mL streptomycin (Solarbio, Beijing, China). Subsequently, cells were seeded in 96-well plates at a concentration of 3,000 cells per well and cultured in CO2 incubator (Thermo, USA) at 37°C and 5% CO2 with saturating humidity. The culture medium was refresh every 48 h until the fifth day.

Extracted molecule

total RNA

Extraction protocol

The total RNA was harvested using Trizol-LS reagent.
The quantity of total RNA was assessed using an Agilent 2100 Bioanalyzer with Agilent RNA 6000 nano Reagents Part 1 kit (Agilent Technologies, USA). For samples of high-throughput sequencing, the RNAs from three replicates at each differentiation stage were pooled as one RNA sample. For each library, small RNA ranging from 10-45 nt in length was purified by polyacrylamide gel electrophoresis and ligated using proprietary adaptors. The modified small RNA was then reverse-transcribed to cDNA and amplified by PCR. Finally, the libraries were sequenced on a BGIseq-500 sequencing platform.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

BGISEQ-500

Description

micRNA-derived 1d pigeon skeletal muscle satellite cells

Data processing

initial sequence was subjected to a series of stringent filters (such as removing low quality-reads, repeated sequences and adaptor sequences) and the output was called clean data. Filtered sequences were then mapped to pigeon reference genome (Columba livia, ColLiv2, GenBank assembly accession: GCA_001887795.1) with stringent criteria (0 mismatch for full length) using Bowtie software.
Next, mappable reads were extended in the reference genome as predicted miRNA precursors. Only candidate precursors that perfectly matched to known pigeon (Columba livia) mature miRNAs annotated by miRBase (Release 22.0) were identified as known pigeon miRNAs (Kozomara A, et al. 2019). Subsequently, to identify the conserved miRNAs, we performed alignments between remaining candidate precursors and seed sequences of mature miRNAs from chicken (Gallus gallus), zebra finch (Taeniopygia guttata) and other mammals, allowing no mismatch.
Genome_build: Genome database:pigeon genome (assembly GCA_001887795.1); miRbase version: V22.0
Supplementary_files_format_and_content: Identified miRNAs in each library with count reads

Submission date

Mar 08, 2020

Last update date

Mar 09, 2020

Contact name

Xun Wang

E-mail(s)

xunwang@sicau.edu.cn

Phone

+862882707606

Organization name

Sichuan agricultural university