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Sample GSM4367203

Query DataSets for GSM4367203

Status

Public on Jan 27, 2021

Title

MHLBJ1: juvenile Mongolian horse long bone1

Sample type

SRA

Source name

Mongolian horse

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