Novaseq 6000 mgiseq t7 sequencing platform

Under isoflurane deep anesthesia, 6 mice were killed by decapital method, including 3 mice in control group and 3 mice in surgery group. The skull was separated by non-sharp method, the hippocampus were placed on ice bags, and then, the hippocampus were frozen in liquid nitrogen and stored in a −80°C refrigerator. The hippocampus were randomly selected for subsequent sequencing analysis. Total RNA was extracted from mouse hippocampus using TRIzol^® reagent (Magen, Guangzhou, China). Nanodrop ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect the A260/A280 absorbance ratio of RNA samples. An Agilent 4150 Bioanalyzer (Agilent Technologies, CA, USA) was used to measure RIN values of RNA. A PE library was prepared according to ABclonal mrna-SEQ Lib Prep Kit (ABclonal, China) specification. Sequencing was performed using the Illumina Novaseq 6000/MGISEQ-T7 sequencing platform (Illumina Inc., Chicago, IL, USA).

The first fully expanded leaves of seedlings plants grown under control and cold temperature conditions were harvested. Total RNA was extracted from rice leaves using Trizol reagent (Shanghai Thermo Fisher Scientific Co., Ltd., Shanghai, China). The Nanodrop ND-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 4150 (Agilent Technologies, Santa Clara, CA, USA) were used to detect RNA quality and concentration, respectively. The mRNA was purified with oligo (dT) magnetic beads, and the mRNA was fragmented in ABclonal First Strand Synthesis Reaction Buffer. Random primers and reverse transcriptase were used to synthesize the first-strand cDNA. The synthesized cDNA was amplified by polymerase chain reaction (PCR) and sequenced using the Illumina Novaseq 6000/MGISEQ-T7 sequencing platform. Transcriptome sequencing and analytical services were completed by Shanghai Zhongke New Life Biotechnology Co., Ltd. (Shanghai, China).

The qualified RNA was prepared according to the instructions of the AB clonal transcriptomic analysis Lib Perp Kit to prepare the PE library. Library quality was assessed using an Agilent Bioanalyzer 4150 and the Illumina Novaseq 6000/MGISEQ-T7 sequencing platform was applied for sequencing. HISAT2 software (http://daehwankimlab.github.io/hisat2/) was employed to compare the clean reads obtained by processing the Perl script [35 (link)] with the reference genome [36 (link)] to obtain mapped reads, and further FPKM values for each gene were calculated in Feature Counts (http://subread.sourceforge.net/).