Hiseq x ten pe150 platform

Total RNA was isolated using the Trizol reagent (Promega, Madison, WI, USA), after which a Nano Photometer® spectrophotometer (IMPLEN, CA, USA) was used to check for the purity of the RNA. Next, RNA concentration was measured using the Qubit® RNA Assay Kit in a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA), and RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total of 3 μg RNA per sample was used as the input material for the RNA sample preparations. Sequencing libraries were generated using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA) according to manufacturer recommendations. Finally, 30 RNA libraries were constructed with the library quality assessed using the Agilent Bioanalyzer 2100 system. Each cDNA library was sequenced on an Illumina Hiseqxten-PE150 platform. Raw RNA-seq data have been deposited in the Sequence Read Archive (SRA) in NCBI. The BioProject number is PRJNA684971.

The libraries were prepared by following the protocol developed by Qi et al. [60 (link)]. Briefly, genomic DNA was digested using MspI and PstI-HF (NEB) at 37℃ for 2 h, then, to inactivate the restriction enzymes at 75℃ for 20 min. The barcoded PstI-HF adapter and common MspI adapater were respectively ligated on the corresponding restriction sites of all samples by T4 DNA ligase (NEB). Ligation reaction lasted for 2 h at 22℃. Following ligation, fragments less than 300 bp were filtered out. PCR amplification was done for each sample separately. PCR products were checked on a 1.0% agarose gel. Primers, dNTP and small DNA fragments were removed from the pooled DNA. Final libraries were sequenced using Illumina Hiseq Xten, PE150 Platform.

The total RNA extraction kit (TIANGEN) was used to extract total RNA from lymphocytes in strict accordance with the operation steps in the instructions. We obtained mononuclear cells from 2ml peripheral blood using the TRIzol reagent according to the manufacturer's protocol. The concentrations of RNA were evaluated using a NanoDrop ND‐2000 spectrophotometer (Thermo Scientific). A total of 200ng RNA was converted into cDNA by reverse transcription using a Transciptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer's protocol on a T100TM Thermal Cycler (Bio‐Rad Inc).²⁴ Quality inspection was carried out with Agilent2200, and cDNA was stored at −80°C.
Then, the libraries were constructed following the protocol from Liang et al.²⁵ For TCRβ CDR3 library preparation, two‐round nested amplicon arm‐PCR was performed with a Multiplex PCR Assay Kit Ver.2 (TaKaRa) using specific primers against each variable and constant gene. PCR products were purified by agarose gel electrophoresis, amplified using Illumina sequencing primers with different sample barcodes and subjected to high‐throughput sequencing using the Illumina HiSeq X Ten PE150 platform.

Genomic DNA was extracted from young leaves of 16 accessions following a standard CTAB protocol^{69 (link)}. DNA libraries were constructed by Novogene and sequenced with the Illumina Hiseq Xten PE150 platform with an insert size of approximately 500 bp at an average depth of 5.2 (Supplementary Table 1). For each accession, the quality control for raw reads were conducted using Trimmomatic^{70 (link)}. The leading and trailing low quality bases (below quality 3) were removed. Each read was scanned with a 4-base sliding window, cutting when the average quality per base drops below 15. Reads below the 36 bases long were removed.

Approximately 5 mg of tissue from each of the skeletal muscle, spleen, heart, liver, and fat samples was crushed into a fine powder in liquid nitrogen (Supplementary Table 1). Pulverized tissues were suspended in 1 mL ice-cold PBS and then gently spun down. The cell pellet was resuspended in 1 mL lysis buffer (50 mM HEPES with pH 7.5 [Life technologies, 15630-080], 140 mM NaCl [Ambion, AM9760G], 1 mM EDTA with pH 8.0 [Ambion, AM9260G], 10% glycerol [Sigma-Aldrich, G7757], 0.5% NP-40 [Roche, 11754599001], and 0.25% Triton X-100 [Amresco, 0694]), followed by isolation of 50,000 nuclei using a previously published protocol with minor modifications⁷⁹ . Then, the transposition reaction mix (12.5 μL TD buffer, 10 μL ddH₂O, and 2.5 μL TDE [Illumina, FC-121-1030]) was added to the isolated nuclei. The reaction system was incubated at 37 °C for 1 h followed immediately by purification with a QIAGEN MinElute PCR Purification Kit (QIAGEN, 28006). The transposed DNA fragments were amplified with the appropriate number of cycles as determined by qPCR. Size selection of the amplified PCR products (100–600 bp fragments) was performed by gel-purification informing previously studies^{79 –81 (link)}. Products from the size selection step were quantified and sequenced using an Illumina HiSeq X Ten PE150 platform.

Total DNA was fragmented by ultrasonication, and 350 bp DNA fragments were recovered by gel cutting. Libraries with an average length of 350 bp were constructed using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, United States). Sequencing was performed on the HiSeq X Ten PE150 platform (Illumina).