organism: Sus scrofa
description: Protocols: At the age of 180 days post-natum, the pigs were electronarcotized and slaughtered by exsanguination. The pig's brains were immediately removed. Different brain regions of the amygdala, hippocampus and hypothalamus were anatomically localized and excised followed a reference guide described by Felix et.al, 1999 “Stereotaxic atlas of the pig brain” Brain Res. Bull., 49(1):1-137. The experimental procedures have been approved by the Animal Care Committee of the Leibniz Institute for Farm Animal Biology. All tissue samples were flash-frozen in liquid nitrogen and stored at -80C until the RNA isolation. Small RNA was purified using an miRNeasy Mini Kit (Qiagen). Briefly, total RNA was extracted from 30-50 mg of brain tissue samples using the QIAzol lysis reagent (Qiagen). Large RNA fractions were seperated by an RNeasy Mini spin column and a seperate fraction enriched in miRNA and other small RNA species was further purified using an RNeasy MinElute spin column according to the manufracturer's recomendation. Integrity of the total RNA was estimated using an Agilent RNA 6000 Nano kit and the enrichment and quantity of small RNAs including miRNAs were assessed using an Agilent small RNA kit on the 21000 Bioanalyzer system (Agilent). Small RNA library preparation was carried out using TruSeq Small RNA Library Preparation Kit according to the manufracture's recommendation (Illumina). Essentially, 250 ng of small RNA fraction was ligated to the RNA 3´Adapter and then to the RNA 5´Adater, respectively. The adapter- ligated RNA was reverse transcibed to generate cDNA and PCR amplified for 15 cycles to enrich the cDNA library. Each library was size-seperated on 6% polyacrylamide gel. The DNA library fragments of approximately 145 -200 nt were excised from the gel and recovered by a standard ethanol precipitation with glycogen and NaOAc. Quality of the DNA library was assessed on a 2100 Bioanalyzer using the High Sensitive DNA assay (Agilent) and normalized to 2nM using a KAPA library quantification kit (KAPA Biosystem).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-01-03T15:59:15Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-12-07T13:27:25Z
attributes.tag: External Id
attributes.value: SAMEA5158090
attributes.tag: INSDC center name
attributes.value: Leibniz-Institute for Farm Animal Biology (FBN) Institute of Genome Biology/ Genomics Unit
attributes.tag: INSDC first public
attributes.value: 2020-01-03T15:59:15Z
attributes.tag: INSDC last update
attributes.value: 2018-12-07T13:27:25Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7499:Amy2900
attributes.tag: age
attributes.value: 180
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: clinical history
attributes.value: intact
attributes.tag: common name
attributes.value: pig
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: 2900
attributes.tag: organism part
attributes.value: amygdala
attributes.tag: sample name
attributes.value: E-MTAB-7499:Amy2900
attributes.tag: scientific_name
attributes.value: Sus scrofa
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: German Landrace