In this study, an RNA-Seq experiment with 3 individual leaf samples was used to identify RNA editing events. The total RNA was extracted from mature foliage using an Extract kit (RP3301, BioTeke, China). The RNA-Seq library construction and sequencing were performed at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The filtered paired-end reads obtained from an Illumina HiSeq 2000, were aligned to the B. platyphylla cp genome using HISAT2 (v2.1.0) software with strict comparison conditions. SAMtools (v1.9), bedtools (v2.25.0) and ChloroSeq were used to call and analyse precise RNA editing sites [28 ]. Because SNPs or mismatches may interfere with the results, we also mapped the set of PE 100 bp-long reads that was used to assemble the B. platyphylla cp genome back to the cp genome sequence using bowtie2 (v2.3.4.1) software and then checked the SNPs. Finally, we designed several pairs of primers using Primer Premier 6.0 software (PREMIER Biosoft International, Canada) and amplified the target sequence by PCR to form genomic DNA (gDNA) and complementary DNA (cDNA). The target representative editing sites were confirmed by Sanger sequencing. The relevant primer information is summarized in Additional file 1 : Table S2.
Extract kit
Manufactured by BioTeke
Sourced in China
The Extract kit is a laboratory equipment designed for the extraction of biomolecules, such as DNA, RNA, or proteins, from biological samples. It provides the necessary tools and reagents to perform the extraction process efficiently and effectively.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using extract kit
1
RNA Editing Identification in Birch
2
Comparative RNA-Seq Analysis of Populus Tissues
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An RNA-Seq experiment with two biological replicates was conducted to compare gene expression patterns of Qu-2 cells with different tissues of P. trichocarpa. After dark culture for 6 d, the total RNA was extracted using an Extract kit (RP3301, BioTeke, China). RNA-Seq libraries (PE150) were prepared and sequenced on the MGISEQ-2000 platform (Beijing Genomics Institute, Shenzhen, China) at BGI-Wuhan (BGI, Wuhan, China). In addition, other RNA-Seq data from different tissues of P. trichocarpa were downloaded from NCBI SRA (Sequence Read Archive) database (shoot: SRR3472992, SRR3472993; leaf: SRR3472995, SRR3472996; root: SRR3473000, SRR3473001; xylem: SRR3473003, SRR3473005; phloem: SRR3472997, SRR3472998; fiber: SRR3473006, SRR3473008; vessel: SRR3473010, SRR3473011) (Shi et al. 2017; Chen et al. 2019) . Raw data were processed by fastp (v0.19.7) for adapter removal and low-quality read filtering. The qualities of the clean reads were checked using FastQC (v0.11.8). The P. trichocarpa annotated with gene set (v3.0) was downloaded from the Phytozome version 12. We used the kallisto (v0.46.0) (Bray et al. 2016) to calculate gene expression levels and calculated Pearson correlation between Qu-2 cell line protoplasts and other samples.
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