Qiaquick pcr extraction kit

To obtain enough RNA, from PQs, PKs, WMs and WFs samples using TRIzol reagent and checked the total RNA quality assessed an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Three replicates of each caste WMs, WFs, PKs and PQs, were pooled to obtain enough RNA and build cDNA libraries using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB). In brief, using Oligo(dT) beads to extract the total RNA, enriched from mRNA and fragmentation buffer were used to transcribed into short fragments into cDNA with random primers. Second-strand cDNA was synthesized by using DNA polymerase I, RNase H, dNTPs and buffer. The cDNA fragments were then purified with the QiaQuick PCR extraction kit, poly(A) end-repaired was tailed, and attached to the Illumina sequencing adapters. Ligation products were selected in size by agarose gel electrophoresis, PCR amplified and sequenced using the Illumina HiSeqTM 4000 platform by Gene Denovo Biotechnology Co. (Guangzhou, China)^{66 (link),67} .

RNA isolation and sequencing were operated according to the reference^{40 (link)}, with some modifications. Briefly, total RNA was extracted from CB and NS using Vazyme-innovation in enzyme technology reagent following the manufacturer’s instructions. Next, mRNA was purified from total RNA using Oligo (dT) beads, fragmented into short fragments using fragmentation buffer and reversely transcribed into first-strand cDNA with random primers. The second-strand cDNA was synthesized by DNA polymerase I, RNase H, dNTPs and buffer. Subsequently, the cDNA fragments were purified with QiaQuick PCR extraction kit, end repaired, poly (A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, amplified by PCR, and sequenced using Illumina HiSeqTM4000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

G. uralensis leaves treated with UV-B irradiation for 6, 12, 24, 48, and 96 h, as well as the control, were collected for RNA extraction. Each treatment was repeated with three biological replicates. Total RNA was extracted from seedlings using an RNAprep Pure Plant kit (Tiangen, China) according to the manufacturer’s instructions. After enrichment with oligo(dT) beads, the fragmented mRNA was reverse transcribed into cDNA with random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTP, and buffer. The cDNA fragments were purified with a QiaQuick PCR Extraction kit, end repaired, poly(A) tailed, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using an Illumina HiSeq. 4000 system from Gene Denovo Biotechnology Co. (Guangzhou, China). The metrics of RNA sequencing analysis are provided in Supplementary Table S7.

The taproots of one-year-old P. notoginseng plants collected in March, May, July, and November were used for RNA-Seq. Each sampling point had three biological replicates. After the total RNA was extracted, the eukaryotic mRNA was enriched by Oligo (dT) beads, while prokaryotic mRNA was enriched by removing rRNA by a Ribo-ZeroTM Magnetic Kit (Epicentre). The enriched mRNA was fragmented into short fragments using fragmentation buffer and then reverse transcribed into cDNA with random primers. Second-strand cDNA was synthesized by DNA polymerase I, RNase H, dNTPs, and buffer. The cDNA fragments were then purified using a QiaQuick PCR extraction kit, and following end-repair and the addition of poly(A) the fragments were ligated to the Illumina sequencing adapters. The ligation products were selected by agarose gel electrophoresis, PCR-amplified, and then sequenced using the Illumina HiSeqTM 4000 at Gene Denovo Biotechnology Co. (Guangzhou, China).

Newly emerged leaves from wild type and rubescent mutants were collected and total RNA was isolated using the cetyltrimethylammonium bromide (CTAB) method (Wang et al., 2011 (link)). Two cDNA libraries each for the wild type and rubescent mutant were prepared for 100 bp paired-end RNA-Seq transcriptome. cDNA library construction and sequencing analysis were performed by Hangzhou Woosen Biotechnology Co. Ltd (Hangzhou, Zhejiang, China).
Poly (A) mRNA was purified with oligo (dT) beads from total RNA, and then the mRNA-enriched RNA was randomly segmented into 200–700 nt fragments in a divalent cation fragmentation buffer (Illumina, Hayward, CA) for 8 min at 94°C. These short fragments were used as templates to synthesize the first-strand cDNA using random hexamer primers and the second-strand cDNA was generated using RNaseH and DNA polymerase I. Those short cDNA fragments were purified with QiaQuick PCR extraction kit and washed with elution buffer (EB) for end repairing and tailing-A. Then those short fragments were ligated to sequencing adapters according to Illumina's protocol (San Diego, CA, USA) and further separated by agarose gel electrophoresis. Fragments of 300–500 bp were enriched by PCR amplification to create a cDNA library.

Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from the flower buds of SXCMS5A and SXCMS5B. The construction of cDNA library referred to prokaryote, considering that the plant mitochondrial genomes were similar to its ring genome. So, after total RNA was extracted, sample mRNA was enriched by removing rRNA by a Ribo-ZeroTM Magnetic Kit (Epicenter, Madison, WI, USA). Next, the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse-transcribed into cDNA with random primers. Second-strand cDNA was synthesized with DNA polymerase I, RNase H, dNTPs, and buffer. The cDNA fragments were then purified with a QiaQuick PCR extraction kit, end-repaired, poly(A) was added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeqTM 2500 by Gene Denovo Biotechnology Co. (Guangzhou, China).