Novaseq machine

mESCs cells were seeded and controlled to afford the same amounts of cells. After 48 h, 5-ethynyl uridine (EU) was added to 0.5 mmol/L at 60, 30, 20, and 10 min before trypsinization collection. Total RNA was purified by Trizol reagent, and nascent RNA was captured by Click-iT Nascent RNA capture Kit. ERCC RNA spike-in control (Ambion) was added to each sample (0.01 µL per sample) before constructing the library with SMARTer StarstarTotal RNA-Seq Kit V2. Sequencing was performed at the University of Chicago Genomics Facility on an Illumina NovaSeq machine.

PCR amplification was conducted using bacteria-specific primers targeting the 16S rRNA V3+V4 region. The primer sequences used were as follows: Forward primer 338F (5′-barcode + ACTCCTACGGGAGGCAGCA-3′) and reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The template DNA was pre-denatured at 98°C for 30 s using the PCR instrument, ensuring its completed denaturation for the subsequent amplification cycle. Within each cycle, the template was subjected to a 15-s denaturation step at 98°C, followed by a 30-s primer annealing step at 50°C to ensure optimal primer-template binding. The temperature was then maintained at 72°C for 30 s, facilitating primer extension, DNA synthesis, and completion of a single cycle. This cycle was iterated between 25 an 27 times to accumulate a substantial quantity of amplified DNA fragments. Finally, the product was kept at 72°C for 5 min so that it was completely extended and preserved at 4°C. The amplification results were subjected to 2% agarose gel electrophoresis, and the target fragments were cut out, and then, the target fragments were recovered using the Axygen gel recovery kit. Then, 2 × 250 bp double-ended sequencing was performed on the Illumina NovaSeq machine using the Novaseq 6000 SP Reagent Kit (500 cycles). Sample DNA extraction, amplification, and library sequencing were completed by Shanghai Personalbio Technology Co., Ltd.

Total RNA was isolated from the soluble nuclear fraction/nucleoplasm fraction of mESCs. Then the mRNA was enriched from total RNA by using the Dynabeads mRNA DIRECT™ kit. SMARTer Stranded Total RNA-seq Kit v2 (Takara) were used to prepare the library according to the manufacturer’s instructions. Sequencing was performed at the University of Chicago Genomics Facility on an Illumina NovaSeq machine.

All 27 P. graminis‐infected leaf and stem samples were subjected to RNA extraction using an RNeasy Plant Mini Kit (QIAGEN) and the quality and quantity of extracted RNA assessed using an Agilent 2100 Bioanalyzer. Genewiz constructed complementary DNA libraries using the TruSeq RNA Sample Preparation Kit (Illumina) and sequenced these libraries on an Illumina NovaSeq machine, generating 150 bp paired‐end reads. Adapter and barcode trimming, and quality filtering were performed on the paired‐end reads using FASTX‐Toolkit v. 0.0.13.2.

mRNA was enriched from total RNA by using the Dynabeads mRNA purification kit. SMARTer Stranded Total RNA-seq Kit v2 were used to prepare the library according to the manufacturer’s instructions. Sequencing was performed at Berry Genomics (China) on an Illumina NovaSeq machine.

Total RNA was extracted from snap-frozen pellets of CBO at day 25, 50, 100, 150, 200, fetal cortical progenitors and fetal brain tissues using the Rneasy Mini Kit (Qiagen, 74104). Purified RNA was quantified using a NanoDrop spectrophotometer and RNA quality was checked with an Agilent 2100 Bioanalyzer using the RNA nano kit (Agilent, 5067-1512). Library preparation for RNA sequencing was performed according to TruSeq Total RNA sample preparation protocol (Illumina, RS-122-2202), starting from 250 ng to 1 μg of total RNA. cDNA library quality was assessed with an Agilent 2100 Bioanalyzer, using the high-sensitivity DNA kit (Agilent 5067-4626). Libraries were sequenced with the Illumina Novaseq machine at a read length of 50 bp paired-end and a coverage of 35 million of reads per sample.