Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), after which the concentration, quality and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). Three micrograms of RNA were ud as input material for the RNA sample preparations. Sequencing libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesize dusing random oligonucleotides and SuperScript II. Second strand cDNA synthesis was subquently performed using DNA Polymera I and RNa H. Remaining overhangs were converted into blunt ends via exonuclea/polymera activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To lect cDNA fragments of the preferred 200 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were lectively enriche
东北鼢鼠d using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high nsitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The quencing library was then quenced on a Hiq platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd.
1.根据您的结题报告，当时是用无参进行的该项测序，这边您只需说明即可。
3.牛排英语关于Trinity的描述：De novo transcriptome asmbly was performed using Trinity (trinityrnaq.sf) . A K-mer library was constructed with the filtered reads, and the contigs were formed using Inchworm. Using Chrysalis, a component was built with the contigs, and de Bruijn graphs were generated. Then, Butterfly was ud to optimize the de Bruijn graphs and create the final transcript through paths。
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这边您可以借鉴。（同问题7，转录本的组装）
人类英雄4.关于GO和KEGG粗浅的分析方法：Blast2go program was ud to annotate the unigenes on the basis of GO terms and NR databa annotation. Conrvation of gene identities of ot
her species was analyzed using BLASTX. To annotate genes with common denominators or functional categories, the unigenes were also aligned to the eggNOG databa (, ). To summarize the pathway information, the KEGG Automatic AnnotationServer（KAAS）was ud to perform pathway annotation.您可以在此基础上把您的比对数据及显著性情况加入。（同问题8）
5.差异基因的筛选标准：我们是以foldchange＞2， p<0.05为显著上调，foldchange<0.5 ,p<0.05为显著下调，您可以在此基础上筛选差异基因做验证，具体的筛选标准您可以附上。
10. 您可以在文章中把我们分析到的不同GO和KEGG桀骜2021年考研国家线聚类下的基因分别列出其功能和通路信息。腌菜花
