High throughput sequencing platform hiseq miseq

One milliliter of TRIzol reagent was added to each vial of cells to extract total RNA from the cells. RNA integrity was accurately detected using an Agilent 2100 RNA Nano 6000 Assay Kit (Agilent Technologies, CA, USA). Three micrograms of total RNA per sample was used as the starting material for library construction, and upon completion, initial quantification was performed using Qubit 3.0. Agilent 2100 for detection. Bio-Rad KIT iQ SYBR GRN was used for Q-PCR. The effective library concentration was accurately quantified (effective library concentration > 10 nM). The different libraries were pooled into a flow cell according to the effective concentration and the target downstream data volume needed. cBOT was clustered and sequenced using the Illumina high-throughput sequencing platform (HiSeq/MiSeq).

Total RNA was extracted from STH and STH×SFK (n = 6 for each group) sheep longissimus dorsi muscle using Trizol reagent. After ensuring quality control, the longissimus dorsi muscle mRNA was enriched with magnetic beads with Oligo (dT) and a strand of cDNA was randomly synthesized using the mRNA as a template; complementary two-stranded cDNA was synthesized, double-stranded cDNA was purified, end repair was performed, sequence adapters were used, and AMPure XP beads (Beckman Coulter, Beverly, MA, USA) were used for fragment size selection (length, 200–500 bp). Finally, PCR amplification was performed to construct the cDNA library. After the library was validated, the Illumina high-throughput sequencing platform (HiSeq/MiSeq) was used for sequencing. Transcriptome sequencing was performed by the Guangzhou Saizhe Biological Company (Guangzhou, China).

The experimental samples we collected were the fresh leaves of T. nigrans and S. parasitica, that parasitize on Platanus acerifolia and Ligustrum lucidum, respectively. The samples were collected at Sichuan University. We immediately froze those leaves in a liquid nitrogen tank and sent the frozen leaves to the Novogene Company for DNA extraction and genome sequencing. The modified CTAB method [27 ] was used to extract the total DNA of the leaf samples and Nanodrop was applied to detect DNA purity (OD 260/280 ratio). Sequencing was performed using the Illumina High-throughput Sequencing Platform (HiSeq/MiSeq). Finally, we obtained raw reads of 6.73 G and 6.85 G from T. nigrans and S. parasitica samples, respectively, and their GC contents were 42.3% and 41.6%, respectively.
We used the Trimmomatic v0.36 [28 (link)] to filter the raw reads and obtain high-quality clean reads. The BWA-MEM V0.7.12 [29 (link)] was used to compare the clean reads using T. chinensis as the reference chloroplast genome (Genbank: NC_036306.1). The read sequence was mapped to the corresponding reference genome. We used NOVOPlasty v2.6.3 [30 (link)] and Velvet v1.2.07 [31 (link)] to assemble and splice chloroplast genomes. We spliced contigs into scaffold sequences and then used them to assemble the chloroplast genome.