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Sample GSM4286960

Query DataSets for GSM4286960

Status

Public on Aug 26, 2020

Title

12-month-B1

Sample type

SRA

Source name

submandibular gland

Organism

Capra hircus

Characteristics

age: 12-month

Extracted molecule

total RNA

Extraction protocol

After slaughter and exsanguination, the submandibular gland was collected, placed in liquid nitrogen immediately and then stored at −80 °C for long-term storage. Total RNA was extracted from 9 submandibular gland samples using TRIzol (Invitrogen, Carlsbad, CA, USA) reagent. The integrity and quality of the extracted RNA was determined by measuring the absorbance at 260/280 nm (A260/A280) using an Agilent BioAnalyzer 2100 (Agilent, Shanghai, China).
Ribosomal RNA was removed, and all other RNA was retained. Total RNA was randomly cleaved into short fragments, and to synthesize first-strand cDNA, the fragmented RNA was used as the template, with random hexamers, buffered dNTPs (dUTP instead of dTTP) and RnaseH. Second-strand cDNA was synthesized using DNA polymerase I. cDNAs were purified using a QiaQuick PCR kit (QIAGEN) and eluted with EB buffer (10 mM Tris-HCl, pH 8.0). The cohesive end was repaired, and an adaptor sequence was ligated to base “A” at the 3’end of the cDNA; the second strand was subsequently degraded with uracil-N-glycosylase (UNG). The ligated product was reverse transcribed by PCR amplification, and the product was purified to generate a cDNA library. To generate the library, the cDNA was sequenced using an Illumina HiSeqTM 4000 from Gene Denovo Biotechnology Co. (Guangzhou, China).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

The sequence containing the adaptor was removed . Reads less than 50 nt after cleaving the adaptor sequences were discarded. Reads that were all A bases were removed. Reads with an N ratio greater than 10% were removed. Low-quality reads were removed (number of bases with a quality score (Q value) greater than 50% of the total reads).
The alignment tool Bowtie2 (2.2.8) was used to map the high-quality clean reads to a ribosomal RNA (rRNA) database (mismatch number: 0). rRNA reads were removed, and then the comparison software Tophat2 (2.1.1) was used to align the remaining reads to the goat reference genome
The tophat2-based alignment results were used to reconstruct transcripts using Cufflinks.
Genome_build: Capra hircus ARS1
Supplementary_files_format_and_content: Excel table file with raw counts of each sample

Submission date

Jan 28, 2020

Last update date

Aug 26, 2020

Contact name

Chao Tianle

E-mail(s)

chaotianle@sdau.edu.cn

Phone

15153876133

Organization name

6/5000 Shandong Agricultural University