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Pipeline publication

[…] p fragments. The cDNA was synthesized using a random hexamer primer and purified with magnetic beads. After the end reparation and 3ˈ end single nucleotide acid addition, the adaptors were ligated to the fragments. The fragments were enriched through PCR amplification and purified using magnetic beads. The libraries were assessed using the Agilent 2100 Bioanalyzer and quantified using the ABI StepOnePlus Real-Time PCR System. The samples were sequenced on an Illumina HiSeq 2000 with paired ends (BGI Tech, Shenzhen, China)., Low-quality reads with Phred scores < 20 were trimmed using Fastq_clean [], and the data quality was assessed using FASTQC []. The filtered reads were assembled using Trinity (version 2.0.6) with default parameters [, ]. The paired-end reads from each library were mapped to de novo assemblies using bowtie (version 1.1.1) []. The transcript abundance was estimated using Corset (version 1.03) []. The count data generated from Corset were processed using the edgeR package []. Transcripts with less than one count per million reads (CPM) for at least three libraries were removed, and the remaining data were used for the next analysis. A matrix was constructed using the single factor style. Effective library sizes were determined using the trimmed mean of M values (TMM) normalization method. The common dispersion and tag wise dispersion were estimated using the quantile-adjusted conditional maximum likelihood (qCML) method. The exact test was performed to compute the expression of genes between the treatment and mock groups. Raw P values were adjusted for multiple testing using a false discovery rate (FDR) []. Genes with an FDR of less than 0.05 an […]

Pipeline specifications

Software tools Trinity, Bowtie, Corset, edgeR