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Total RNA was extracted from wild-type and Zmgrp23-mu/Zmgrp23-ems mutant endosperms using the RNeasy Plant Mini Kit (Qiagen, Germany), followed by DNase I (NEB, USA) digestion to eliminate genomic DNA contamination. First-strand cDNA was synthesized using mitochondrial and chloroplast gene-specific primers with SuperScript II Reverse Transcriptase (Invitrogen, USA). Target genes were amplified via PCR using the same primers. The PCR products were validated by agarose gel electrophoresis, pooled in equimolar concentrations, and fragmented to ~350 bp using a Covaris M220 Focused-ultrasonicator (50W peak power, 10% duty cycle, 90s treatment). Sequencing libraries were prepared with the Truseq Nano DNA HT Kit (Illumina) and subjected to paired-end 150 bp sequencing (insert size ~350 bp) on the Illumina NovaSeq platform. |
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RT-PCR |
PAIRED
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