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[…] ubated with DNase I (Ambion, USA), and messenger RNA was further purified with MicroPoly(A) Purist Kit (Ambion, USA) as per the protocol and the final concentration was determined using NonoDrop., RNA was fragmented and annealed with Biotinylated Random Primers which have the Illumina adapter sequence. Then the RNA fragments were captured by Strapavidin through Biotinylated Random Primers. Another Illumina adapter was ligased to 5′RNA by RNA ligase. Reverse transcriptase was used for reverse transcription. Finally two double strand Illumina libraries were obtained by PCR amplification., The two libraries were sequenced by Illumina paired-end sequencing technology. Raw data was scanned using Casava with default parameters, and reads with more 10% Q<20 bases were removed. All sequences smaller than 70 bases were eliminated based on the assumption that small reads might represent sequencing artifacts . Then the high quality reads were assembled by Trinity with default parameters to construct unique consensus sequences ., After de novo assembled with Trinity, open reading frames were identified by using an in-house developed program based on ‘GetORF’ from EMBOSS . Gene annotation was performed through BLASTp search against Swiss-Prot and GenBank database with E value of 10−5, and then the best one was chosen as the result of gene annotation. Comparisons with the ESTs of Capsicum annuum in NCBI and tomato CDS in SGN were performed through Perl scripts, with E value less than 10−10 and 10−20, respectively, and the proportion of the similar part larger than 80%. Gene ontology analysis was performed using GoPipe , BLASTP was firstly used to search against Swiss-Prot and TrEMBL database with E value of 10−5, and then the GO information was obtained according to gene2go. The metabolic pathways were constructed based on KEGG database by BBH (bi-directional best hit) method . KO number of each protein was identified firstly and metabolic pathways were constructed based on the KO number then., Reads number of each unigene was firstly transformed into RPKM (Reads per Kilo bases per Million reads) and then differently expressed unigenes were identified by DEGseq package using the method MARS (MA-plot-based method with Random Sampling model) . “FDR ≤0.001 and the absolute value of log2Ratio ≥1” was used as the threshold to judge the significance of unigene expression difference. The data analyzed have been deposited on the NCBI Gene Expression Omnibus under accession no. GSE45431., Candidate male fertility-related genes were selected according to their annotation (), and then their abundance differentiations of the two materials were obtained from the result of gene expression analysis ()., All unigenes showing significant transcript abundance differences between the two materials were firstly mapped to the GO and KEGG pathway databases, and t […]

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