E z n a kit

P7 4-OHT treated primary neurospheres were pooled and dissociated as in [67 (link)]. Resulting neurosphere cells were cultured at clonal density (2 cells/μl) as secondary neurospheres as described in [67 (link)] for 7 DIV in a 6-well polystyrene microplate with 2 ml of SFM supplemented with FGF, EGF, B27, and heparin. Secondary neurospheres were counted and then centrifuged at 465 g for 7 min at room temperature, and the cell pellet was collected for total RNA purification according to manufacturer’s instructions (Omega Bio-Tek E.Z.N.A kit).

According to the operation instructions, E.Z.N.A.™ Kit (Omega Bio-Tek, Norcross, GA, USA) was applied to extract the total RNA from the DI flora. Gene Denovo Co., Ltd. (Guangzhou, China) assisted in sequence detection and data analysis. The original data were recorded in the NCBI SRA, with the accession number PRJNA666309. The Supplementary File included specified analysis procedures.

Total RNA was extracted by using the E.Z.N.A. Kit (OMEGA Bio-Tek, Norcross, GA, USA) from frozen plant material according to the manufacturer's instructions. RNA integrity was visualized in 1% agarose gels while concentration and purity (260/280 ratio) was determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA was treated with DNase I (Fermentas, Hanover, MD, USA) to remove DNA contamination. First-strand cDNA was carried out using 2 μg total RNA with the PrimerScript RT-PCR kit from Takara (Condalab, Barcelona, Spain) following the manufacture's protocol.

Fresh fecal samples were collected at 19 weeks of age, and the samples were subsequently transferred to dry ice and stored at −80 °C. Microbial DNA was extracted using an EZNA® kit (Omega Bio-Tek, Norcross, GA, USA). A PCR thermocycler system (GeneAmp 9700, ABI, USA) was used to amplify the V3–V4 variable region (primers 338F and 806R), after which the products were extracted from 2% agarose gels using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union, CA, USA). Further purification and quantification were conducted using QuantiFluor™-ST (Promega, USA) according to the manufacturer’s protocol. The GREENGENES database was used for microbiota annotation. LEfSe was used to identify bacteria that differed in abundance between the samples.^{52 (link)} The core diversity plug-in of QIIME2 was used to calculate diversity metrics and the alpha diversity index. The Bray‒Curtis distance was used as an index to explore beta diversity, which was determined via PCoA.^{53 (link)}

The strain MF180151 was isolated from a marine sediment sample from the Bohai Sea, China. It was incubated on potato dextrose agar (PDA) plate consisting (0.4% potato starch, 2% dextrose, and 2% agar) at 28 °C. The identification was performed based on the morphology and phylogenetic analysis. The whole genomic DNA of the strain was extracted using the E.Z.N.A. kit (Omega Bio-Tek, Norcross, GA, USA). A pair of primers (ITS4: 5″-TCCTCCGCTTATTGATATGC-3″; ITS5: 5″-GGAAGTAAAAGTCGTAACAAGG-3″) was used to amplify the ITS region of MF180151. PCR amplification (50.0 μL final volume: 25 μL 2 × Taq Master Mix, 2 μL of 10 μM of each primer, 5.0 μL DNA template and 16 μL ddH₂O) of the ITS sequence was performed on Bio-gener PCR Thermal Cycler with the initial denaturation at 95 °C for 3 min, 32 cycles of denaturation (94 °C, 15 s), annealing (60 °C, 15 s), and elongation (72 °C, 60 s), and a final elongation at 72 °C for 5 min. After multiple alignments of ITS sequence of the related species by CLUSTAL W [37 (link)], phylogenetic analysis was constructed using neighbor-joining method with bootstrap values based on 1000 replications by MEGA 5.0 [38 (link),39 (link)].
The strain was deposited at the Institute of Microbiology, Chinese Academy of Sciences. The nucleotide sequences of ITS gene (accession number MK680178) of A. versicolor MF180151 were deposited in GenBank.