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Protocol publication

[…] uenced on Illumina Hiseq 2000 platform., Through base calling, the original image data of sequencing was analyzed into the sequenced reads, they were called the raw reads. In order to obtain the clean reads, we filtered out the reads of low-quality, and the reads with the percentage of N more than 10%. In addition, reads were removed with 5′ primer contaminants or without 3′ primer insert fragments. After trimming the 3′ adaptors, removing the reads including polyA, poly T, poly C, and poly G, the clean reads were mapped duck genome database (BGI duck 1.0 reference Annotation Release 101) and compared with the distribution of the known miRNAs. The novel miRNAs were predicted by the software miREvo and mirdeep2 [,]. With the statistics and normalization of the expression of miRNAs in two samples through transcript per million (TPM, normalization expression = readCount×1,000,000)/libsize) [], the differential expression of miRNAs were compared by DGEseq in the two samples []. The 0.05 of p-value and log2 Fold-change (log2[JCH/NH-P]) were calculated to filter the significant differential expressions. GOseq Release 2.12 was used to analyze the gene ontology (GO) enrichment of differential expressed miRNAs. According to the relationship between miRNAs and target genes, we analyzed the distribution of target genes in GO through three categories (cellular component, biological process, molecular function). KEGG is the main database of pathway for understanding the high-level functions and utilities of biological system (http://www.genome.jp/kegg/). We used KOBAS v2.0 software to calculate the expression of the miRNA target genes in order to find which pathway was involved., The total RNA were reversed transcribed into first-strand cDNA according to the manufacturer’s instructions of miRcute miRNA reverse transcription kit (TIANGEN, Beijing, China). Some significantly differentially expressed miRNAs were selected to verify by real-time PCR. The mature sequence of miRNAs and information of primers are listed in . U6 was used as the housekeeper genes. The miRcute miRNA RT-PCT kit (TIANGEN, China) was used to determine miRNA expression, which was carried out using an iCycler IQ5 Multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA […]

Pipeline specifications

Software tools miREvo, miRDeep, GOseq, KOBAS
Organisms Anas platyrhynchos