Hiseq control software olb gapipeline 1

Total RNA of each sample was extracted using a TRIzol Reagent (Invitrogen)/RNeasy Mini Kit (Qiagen)/other kits. Total RNA of each sample was quantified and qualified by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. Quantity of 1 μg total RNA with RIN value above 7 was used for the following library preparation. Next generation sequencing library preparations were constructed according to the manufacturer’s protocol (NEBNext® Ultra™ RNA Library Prep Kit for Illumina®). In brief, the PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2 × 150 bp paired-end (PE) configuration; image analysis and base calling were conducted by the HiSeq Control Software (HCS)+OLB+GAPipeline-1.6 (Illumina) on the HiSeq instrument. The sequences were processed and analyzed by GENEWIZ. The detailed information was uploaded to ArrayExpress with accession E-MTAB-6346.

In beif, total RNA of each sample was extracted using TRIzol Reagent (Invitrogen)/RNeasy Mini Kit (Qiagen)/other kits. Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and 1% agrose gel. 1 μg total RNA with RIN value above 7 was used for following library preparation. Next generation sequencing library preparations were constructed according to the manufacturer's protocol (NEBNext^® Ultra^™ RNA Library Prep Kit for Illumina^®). Sequencing was carried out using a 2 × 150 bp paired-end (PE) configuration; image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument. The sequences were processed and analyzed by GENEWIZ INC (Suzhou, China).

Five samples from each group were used to extract RNA using HiPure Plant RNA Mini Kit (Magen, Guangzhou, China). The quality was evaluated by NanoDropTMOneC (Thermo Fisher, Waltham, MA, USA). After extraction, 20 µL of RNA samples were transferred into RNA stabilization tubes (GENEWIZ from Azenta Life Sciences, Suzhou, China) and air-dried under the biosafety hood for 24 h. The stabilized RNA samples were shipped to Azenta Life Sciences (Suzhou, China) for sequencing.
Sequencing was carried out using a 2 × 150 bp paired-end (PE) configuration; image analysis and base calling were conducted by the Hiseq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina, San Diego, CA, USA) on the HiSeq instrument. A total of 10 buffel grass seedling samples (n = 5) from both control and phytogenic treatment were successfully extracted and sequenced. All sequence data are accessible on the NCBI Sequenced Read Archive (SRA) database with the accession number PRJNA1068799.

The genome of D-G6 was sequenced by GENEWIZ incorporated company (Shenzhen, China). One hundred nanograms of D-G6 genomic DNA was randomly fragmented to <500 bp via sonication (Covaris S220). The fragments were modified via 5′ phosphorylation and dA-tailing. Next, adaptors were added to both ends after the fragments. Fragments of ~470 bp were purified by size based selection of adaptor-ligated DNA. The library was amplified via PCR for 8 cycles using primers P5 and P7. Following clean up and validation using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and quantified by Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA), sequencing was carried out using a 2 × 150 paired-end (PE) configuration and monitored by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) (San Diego, CA, USA) on a HiSeq instrument and processed via image analysis and base calling.
For analysis performed by Pacbio (Menlo Park, CA, USA), the construction of the SMRTbell library required 10 kb double-stranded DNA fragments end repaired and ligated with universal hairpin adapters. The library was sequenced in a PacBio RSII/Sequel SMRT instrument [48 (link)]. PacBio reads were assembled using HGAP4/Falcon WGS-Assembler 8.2 [49 (link)], and re-corrected through either Pilon software using previous Illumina data or Quiver using Pacbio reads.

RNA sequencing was performed by Illumina HiSe, and total RNA from each sample was extracted using TRIzol Reagent (Invitrogen)/RNeasy Mini Kit (Qiagen)/other kits for the preparation of the following libraries. The PCR products were washed with beads, validated with Qsep100 (Bioptic, Taiwan, China), and quantified with a Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Libraries of different indices were multiplexed and loaded on an Illumina HiSeq instrument (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Sequencing was performed using a 2x150bp paired-end (PE) configuration; image analysis and base calling were performed on the HiSeq instrument by HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina). Sequences were processed and analyzed by GENEWIZ. The CIBERSORT analysis used for the subsequent calculation of the abundance and immune score of the 22 immune cells was drawn by ggplot. RNA-seq data have been deposited in NCBI database (accession code PRJNA724048).