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Sample GSM1656400

Query DataSets for GSM1656400

Status

Public on Dec 05, 2017

Title

B0646

Sample type

SRA

Source name

Endometrium

Organism

Bos indicus

Characteristics

developmental stage: multiparous cows
health status: Healthy
breed: Nelore
treatment: Estradiol cypionate supplemented
pregnant status: Non-pregnant
progesterone (ng/ml) at sample collection: 4.73
tissue: endometrium

Treatment protocol

A total of 12 suckled cows presenting absence of corpus luteum detected by ultrasound received 2 mg of estradiol benzoate im and an intravaginal P4 device. Eight days later, P4 devices were removed, and the cows received 500 mg of sodium cloprostenol IM. Cows were blocked according to body condition score and the diameter of largest follicle (LF) at P4 device removal and were randomly assigned to receive an im treatment with 1mg of ECP (ECP, n=6) or not (CON, n=6) at the P4 device removal. All cows received 10μg of buserelin acetate 48 h after the P4 device removal and were timed artificially inseminated immediately after. Cows presenting a new corpus luteum formed 6 d after the GnRH treatment had an endometrial fragment collected by transcervical biopsy

Extracted molecule

total RNA

Extraction protocol

Approximately 30 mg of endometrial tissue were ground in liquid nitrogen using a stainless steel apparatus and immediately mixed with buffer RLT from the RNeasy Mini columns kit (Qiagen, São Paulo, SP, Brazil), as per manufacturer’s instructions. To maximize lysis, tissue suspension was passed at least ten times through a 21 G needle, and centrifuged at 13,000 x g for 3 minutes for removal of debris, prior to supernatant loading and processing in RNeasy columns. Columns were eluted with 40 l of RNase free water, and elution was repeated using the same 40 l initially used to increase RNA concentration. Concentration of total RNA on extracts was measured by a spectrophotometer (NanoDrop, Thermo Scientific, Wilmington, USA). Prior to reverse-transcription, 1 g of total RNA was treated with DNAse I (Life Technologies, São Paulo, SP, Brazil) for 15 minutes at room temperature in a 10 l reaction, followed by addition of 1l of EDTA (25mM) and heating at 65C for 10 minutes to inactivate DNase I. DNase I treatment was immediately followed by reverse-transcription (High Capacity cDNA Reverse Transcription Kit, Life Technologies) according to manufacturer’s instructions. Briefly, 9 l of master mix containing RT buffer, dNTP mix, random primers, RNase inhibitor and reverse transcriptase were added to the 11 l of DNase I treatment reaction. Immediately, samples were incubated at 25 C for 10 minutes, followed by incubation at 37 C for 2 hours and reverse-transcriptase inactivation at 85C for 5 minutes and storage at -20C.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiScanSQ

Data processing

CASAVA v1.8.2
The 100bp paired end (PE) reads were filtered using a perl script which removed all reads with a mean quality under 26. The reads were mapped with Bowtie2 v2.1.0 on the masked bovine genome assembly (Bos taurus UMD 3.1, NCBI).
The mapping file was sorted using SAMTools v 0.1.18 and read counts were obtained using the script from HTSeq-count v0.5.4p2 (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html).
The differential expression analysis was performed with package DESEq v1.12.1, from R. Using the function estimateSizeFactors, the normalized counts were obtained (baseMean values, which are the number of reads divided by the size factor or normalization constant). The standard deviation along the baseMean values was also calculated for each transcript.
In order to avoid artifacts caused by low expression profiles and high expression variance, only transcripts that had an average of baseMean > 5 and the mean greater than the standard variation were analyzed. The threshold for evaluating significance was obtained by applying a p-value of 0.05 FDR-Benjamini-Hochberg.
Genome_build: Bos taurus (assembly Bos_taurus_UMD_3.1.1)

Submission date

Apr 13, 2015

Last update date

May 15, 2019

Contact name

Mario Binelli

E-mail(s)

binelli@usp.br

Phone

19998591388

Organization name

University of Sao Paulo (USP)