GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM7781201

Query DataSets for GSM7781201

Status

Public on Oct 01, 2023

Title

DP1

Sample type

SRA

Source name

mammary gland

Organism

Capra hircus

Characteristics

tissue: mammary gland
cell line: mammary cell
cell type: dry period
genotype: dry period
treatment: dry period

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using a TRIzol™Reagent Kit (Invitrogen, Carlsbad, CA). RNA concentration and integrity were examined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany), and RNA samples with an RNA integrity number (RIN) > 8 were used for subsequent experiments.
Nine small RNA libraries were established using the total RNA isolated from mammary gland tissue at the LL, DP and LG stages (LL1, LL2, LL3, DP1, DP2, DP3, LG1, LG2 and LG3) with an NEBNext®Ultra™RNA Library Prep Kit (Illumina, San Diego, USA). For each small RNA library, 1 μg of total RNA was subjected to electrophoresis using 12% Tris-borate ethylenediaminetetraacetic acid (TBE)-urea polyacrylamide gel electrophoresis (PAGE) gels (Invitrogen) to obtain small RNA fragments with a length between 18 and 30 nt. The 3’ and 5’ ends of the RNA were ligated with T4 RNA ligase 2 and reverse transcribed to obtain cDNA based on the 3’ and 5’ adapters ligated to the small RNA. The target library was amplified using PCR, and dsDNA fragments between 140 and 160 bp in length were extracted using TBE gel electrophoresis. The libraries were subjected to sequencing using an Illumina/Solexa system after quality tests.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Data processing

The libraries were subjected to sequencing using an Illumina/Solexa system
The data were subjected to quality assessment using FastQC software (v 0.11.9). According to the quality assessment results, the sequencing adapters were processed with Cutadapt software (v 4.2) to eliminate reads with 10% or more unknown bases and a length greater than 30 bp or less than 18 bp and to retain reads with a sequencing quality (Q) > 30.
The filtered clean reads were subjected to sequence comparison with the Silva (https://www.arb-silva.de/), GtRNAdb (http://lowelab.ucsc.edu/GtRNAdb/), Rfam (http://rfam.xfam.org/) and Repbase (https://www.girinst.org/repbase/) databases using Bowtie software (v 1.1.1). Subsequently, the reads were annotated as repeat reads and noncoding RNAs (ncRNAs), including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs) and other RNAs.
The comparison results were summarized, and the short reads were annotated with classifications. Unannotated reads containing miRNAs were obtained after filtering ncRNAs and repeated sequences. The filtered unannotated reads were then aligned with the goat reference genome (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/001/704/415/GCF_001704415.1_ARS1) to determine the locus information on the reference genome. Known miRNAs were identified, and novel miRNAs were predicted with miRDeep2 software using the mature and hairpin sequences of goat, cow and human in the miRBase database (http://www.mirbase.org/).
Transcripts per million (TPM) values of miRNA expression were calculated by miRDeep2 software (v 2.0.1.0). According to the TPM values, miRNAs were divided into four groups: the high-expression group (TPM ≥ 500), medium-expression group (500 > TPM ≥ 10), low-expression group (10 > TPM ≥ 1), and ultra-low-expression group (1 > TPM). The detected known miRNAs and novel miRNAs were subjected to family analysis based on sequence similarity to determine the conservation of miRNAs throughout evolution.
Assembly: GCF_001704415.2
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample
Supplementary files format and content: Matrix table with raw counts for every miRNA and every sample

Submission date

Sep 14, 2023

Last update date

Oct 01, 2023

Contact name

Rong Xuan

E-mail(s)

xuanrong@sdau.edu.cn

Phone

+8618853857973

Organization name

Shandong Agricultural University