GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM4367192 Query DataSets for GSM4367192
Status Public on Jan 27, 2021
Title DPLBJ2: juvenile Debao pony long bone2
Sample type SRA
Source name Debao pony
Organism Equus caballus
Characteristics tissue: epiphysis
developmental stage: juvenile
Sex: female
Extracted molecule total RNA
Extraction protocol Each sample was individually ground (mortar and pestle, under continuous liquid N2 chilling) into a fine powder before RNA extraction. Ground samples were stored at -80°C. Total RNA was extracted from 30 mg of ground tissue by using hot phenol method. In brief, cell pellets were resuspended and washed once in Buffer A (50 mM sodium acetate and 10 mM EDTA, pH=5.2). After collecting the cells by centrifugation, the pellets were resuspended in Buffer A containing 1% SDS and immediately added to hot phenol. After incubation at 65°C for 5 minutes followed by centrifugation for 10 minutes at 4°C, the RNA-containing supernatants were transferred to a new tube for ethanol precipitation, washed and then dissolved in DE PC-treated water. The RNA was further purified with two phenol-chloroform treatments and then treated with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA we redetermined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (BioRad). The integrity of the RNA was further verified by 1.5% agarose gel electrophoresis.
Ribosomal RNAs were removed from the RNA samples (10 μg) using a RiboMinus rRNA depletion kit (Ambion), and the resulting samples we are used to prepare directional RNA-Seq libraries. The purified mRNAs were then iron -fragmented at 95°C followed by end repair and 5' adaptor ligation. Then, reverse transcription was performed using RT primers containing a 3' adaptor sequence and a randomized hexamer. The cDNAs were purified and amplified, and all 200-500-bp PCR product s were purified, quantified and stored at -80°C until they were used for sequencing.
For high-throughput sequencing, libraries were prepared following the manufacturer's instructions, and the Illumina NextSeq 500 was used to collect data from 151 nt paired-end sequencing.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Data processing Illumina bcl2fastq software used for basecalling.
3' adapter (end1:AGATCGGAAGAGC;end2:AGATCGGAAGAGC) and low quality bases trimmed using FASTX-Toolkit (Version 0.0.13). the short reads less than 16nt were also dropped.
Reads were mapped using tophat2 --read-edit-dist 4 -N 4 --b2-N 1 -a 8 -m 0 -g 20 -p 16 --no-discordant. Uniq mapped reads were remained. while several mapped reads have the same start position and end position, only one is considered.
edgeR was used to the DEG analysis with FC>2 Pvalue <= 0.01.
Genome_build: Ensembl, EquCab2.75
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample.
Submission date Mar 01, 2020
Last update date Jan 27, 2021
Contact name Dong Chen
Organization name ABLife, Inc.
Department Center for Genome Analysis
Street address 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone
City Wuhan
State/province Hubei
ZIP/Postal code 430075
Country China
Platform ID GPL21401
Series (1)
GSE146145 Pathways involved in pony body size development
BioSample SAMN14254611
SRA SRX7825274

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap