Sequencing platform

To check the gene expression, RNA was extracted from mouse retina and the cDNA, DNA and small RNA libraries were sequenced on the Illumina sequencing platform by Genedenovo Biotechnology Co., Ltd. (Guangzhou, China). For miRNA-seq, we collected MSC-Exos as previously described, and samples were sequenced on the Illumina sequencing platform by RiboBio Biotechnology Co., Ltd. (Guangzhou, China). For RT-PCR, retinal tissues were harvested immediately using TRIzol reagent (Takara), and total RNAs were extracted using phenol/chloroform, from which 1 mg of RNA was reverse transcribed using the Superscript cDNA kit (Takara). The resulting cDNA was used as a template for subsequent PCR amplification with specific primers (Gene Company Limited). The relative mRNA expression levels of the target gene were normalized to the expression levels of GAPDH determined using the 2^−ΔΔCT method. An All-in-One™ miRNA qRT-PCR Detection Kit 2.0 was used to detect the expression of miR-146a-5p. The expression of miR-146a-5p levels was normalized to the expression levels of U6. The qRT-PCR primers were purchased from Guangzhou GeneCopoeia (Guangzhou, China, http://www.igenebio.com/) and are listed in Additional file 2: Table S1.

The test material was wild walnut seedlings (JF) from Xinjiang, which were planted at the Institute of Horticultural Crops, Xinjiang Academy of Agricultural Sciences, and transplanted into organic matter-rich soil and peat soil (2:1) when the seedlings grew well. When the seedlings grew to five compound leaves, the well-grown plants with consistent growth were screened and placed in an artificial climate chamber (4°C, 16 h light) for low-temperature stress treatment. Three to five leaves under the terminal leaf were taken at 0 h (control) and 8 h of treatment, with three biological replicates for each treatment, and then the leaves were placed in an ultralow-temperature refrigerator at −80°C after quick freezing in liquid nitrogen to carry out transcriptome sequencing via the Illumina sequencing platform. Transcriptome sequencing using the Illumina sequencing platform.
At the same time, well-grown plants with consistent growth were screened and subjected to 4°C low-temperature stress treatment, and 2-5 functional leaves under the parietal leaves and tissue materials of roots, stems, and leaves were removed at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h of treatment, respectively, with three biological replicates for each treatment. The plants were subsequently cleaned and placed in liquid nitrogen quickly frozen and then put into −80°C ultralow-temperature freezers for storage.

Total RNA was isolated from Sf21 cells using Trizol as per manufacturer’s instructions (Life Technologies, USA). The small RNA library was prepared from the total RNA using TrueSeq Small RNA preparation guide (Illumina Inc., USA). Briefly, cDNA was prepared from the total RNA using adapter primers and was resolved on 6% PAGE gel. The gel fragment corresponding to miR population was excised and the library was recovered. Subsequently, the cDNA was analyzed on Agilent Technologies 2100 Bioanalyzer and run on Illumina sequencing platform at University of Delhi South Campus, New Delhi, India.