Ten sample sets used in this experiment that cover six prenatal (entire 114 days of pregnancy) and four postnatal (180 days after birth) developmental stages of Rongchang pigs (a Chinese indigenous breed). Six sows were artificially inseminated with semen from purebred sires and one sow was sacrificed at each of the six prenatal stages (i.e. 30, 45, 60, 75, 90 and 105 days post conception) to obtain three male and three female fetuses. The 30 and 45 days fetuses samples were simply homogenized; other prenatal samples were a mixture of whole body tissues without bones. For four postnatal samples (i.e. birth, 30, 120 and 180 days) the mixture samples were from 60 tissues at equal amounts for each type of tissue. The mixture samples from individual three female and three male fetuses or pigs were kept separately. The low molecular weight RNA was extracted using mirVana miRNA Isolation Kit (Ambion Life Technologies) according to the manufacturer’s protocol. RNA samples were prepared as follows: for each developmental stage, equal quantities (10 µg) of small RNA isolated from six individuals, three female and three male were pooled. Approximately 60 µg of small RNA representing each developmental stage was used for library preparation for sequencing. In brief, the sequencing was performed as follows: small RNA fraction between 18 and 30 nt was isolated by polyacrylamide gel electrophoresis (PAGE), and ligated with proprietary adaptors (Illumina Inc.). The short RNAs were converted to cDNA by RT-PCR and the cDNA was sequenced on the Genome Analyzer GA-I (Illumina Inc.) following the Illumina protocol.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
size fractionation
Instrument model
Illumina Genome Analyzer
Description
Mixture of all tissues, only discard the bones Three female and three male were pooled.
Data processing
Sequ-seqs were processed using the Pipeline software from the Genome Analyzer GA-1 instrument (Illumina Inc.) at first, then were subject to a series of data filtration steps to obtain "mappable sequences", which are sequences satisfy the following criteria from the statistics of miRBbase 13.0: (1) not sequencing adapters; (2) containing no more than 80% A, C, G, or T; (3) containing no more than two N (undermined bases); (4) containing no stretches of A7, C8, G6, or T7; (5) been observed more than two times; (6) being longer than 14 nts and shorter than 27 nts; and (7) not originating from mRNA, rRNA, tRNA, snRNA, snoRNA and repetitive sequence elements. Further data analysis of the "mappable sequences" mainly used ACGT101-miR, our own development software for discovering miRNAs from next-generation sequencing (NGS) data (to be published separately), based on the databases of pig genome (Sscrofa9, April 2009) & EST sequences and mammalian konwn miRNA genes in miRBase 13.0 (March 2009).
