Pepper regeneration capacity analysis

Description

Samples (with description): 94: NonDe (Nondedifferention)；8: ExDe (Excessive dedifferentiation)；19C-1: NorDe (Normal dedifferentiation)；19C-2: AbDi (Abnormal differentiation)；19C-3: NorDi (Normal differentiation). Each sample was repeated 3 times, and each duplicate is represented as: samples name-1/2/3. Abstract (1) The eukaryotic reference transcriptomic (RNA-seq) analysis of 18 samples was completed, and a total of 115.53 Gb of Clean Data was obtained, and the Clean Data of each sample reached 5.70 Gb, and the percentage of Q30 bases was 92.98% or above. (2) The Clean Reads of each sample were sequentially compared with the specified reference genome. Variable splicing prediction analysis, gene structure optimization analysis and discovery of new genes were performed. 18,520 new genes were discovered, of which 14,755 were functional annotation. (3) In this project, Fold Change≥2 and FDR<0.01 were used as differential gene screening criteria.

Steps to reproduce

Total RNA quality was detected using the following methods: (1) NanoDrop 2000 spectrophotometer: detection of RNA purity and concentration; (2) Agient2100/LabChip GX detection: accurate detection of RNA integrity. After the sample test is qualified, the library is constructed. The main process is as follows: (1) Enrichment of eukaryotic mRNA with magnetic beads with Oligo (dT); (2) mRNA was randomly interrupted by Fragmentation Buffer. (3) Using mRNA as template, the first cDNA chain and two strands were synthesized, and the cDNA was purified; (4) The purified double-stranded cDNA was then end-repaired, A-tail was added and sequencing joints were connected, and AMPure XP beads were used for fragment size selection; (5) cDNA library was obtained by PCR enrichment. After the library was constructed, Qubit 3.0 fluorometric quantifier was used for preliminary quantification at a concentration above 1ng/ul, and then Qsep400 high-throughput analysis system was used to detect the inserted fragments of the library. After the inserted fragments met the expectations, The effective concentration of the library (> 2nM) was quantified accurately by Q-PCR to ensure the quality of the library. After the library was qualified, PE150 mode sequencing was performed using Illumina NovaSeq6000 sequencing platform.

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