AP_10-12_5n
Protocols: The insemination of five gilts was performed on days 1-2 of the estrous cycle. Additionally, pregnancy was confirmed by the presence of morphologically normal conceptuses in both uterine horns, and the day of pregnancy was also confirmed by the size and morphology of embryos according to Anderson (1978). The pituitary samples in-tended for further high-throughput transcriptome sequencing, quantitative real-time PCR and DNA capillary sequencing were collected post-mortem from the same ani-mals and at the same time immediately after humanitarian slaughter. Pituitaries were placed in DMEM (Dulbecco's modified Eagle's medium; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 100 IU/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and transported to the laboratory on ice. The pituitary glands were dissected and divided into APs and NPs, and further re-search have been continued only on APs. The anterior lobes were frozen in liquid ni-trogen, and stored at −80 °C until processing for RNA. Total RNA was extracted from all AP samples using RNeasy Mini Kit (Qiagen, Germantown, MD, USA) with DNase contained in RNase-free DNase Kit (Qiagen, Germantown, MD, USA), following manufacturer's protocol. The RNA purity (A260/A280) and quantity (A260) were estimated spectrophotometrically using Infinite M200 Pro (Tecan, Männedorf, Switzerland). The total RNA integrity was validated us-ing Bioanalyzer 2100 (Agilent Technology, St. Clara, CA, USA). An RNA integrity number between 8 and 10 qualifies the isolated RNA for further processing, such as the transcriptome high-throughput sequencing (RNA-Seq) analysis and the quantita-tive real-time polymerase chain reaction (qPCR) validations. Isolated RNA probes were stored at -80 °C for sequencing library preparation. According to Shen's et al. (2016) recommendations the sequencing libraries were prepared by purifying RNA solution from ribosomal RNA with use of Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA). An rRNA-depleted RNA samples were used to prepare strand-specific RNA-Seq libraries using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA), according to manufacturer's procedure. Briefly, long RNA molecules were fragmented, and based on them, double-stranded cDNA molecules were synthesized by replacing dTTPs with dUTPs in reaction solution during the synthesis of the cDNA second strand. The resulting double-stranded cDNA fragments were subjected to the end repair and A-tailing processes. Finally, specific adaptors were ligated into the obtained cDNA fragments. The PCR amplifications was performed to enrich the cDNA libraries.
ENA-FIRST-PUBLIC
2022-12-15
ENA-LAST-UPDATE
2022-12-15
External Id
SAMEA111493104
INSDC center alias
Department of Animal Anatomy and Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn
INSDC center name
department of animal anatomy and physiology, faculty of biology and biotechnology, university of warmia and mazury in olsztyn
INSDC first public
2022-12-15T00:26:22Z
INSDC last update
2022-12-15T00:26:22Z
INSDC status
public
Submitter Id
E-MTAB-12316:AP_10-12_5n
age
7 to 8
broker name
ArrayExpress
common name
pig
developmental stage
adult
genotype
wild type genotype
individual
5
organism part
adenohypophysis
sample name
E-MTAB-12316:AP_10-12_5n
sampling time point
midluteal phase of the estrous cycle
sex
female
strain
Large White × Polish Landrance