Hiseq paired end platform

Harvested spikes from experiments 1 and 2 were used for RNA extraction. Six spikelets with rachis were dissected from the middle of each treated spike and ground up in liquid nitrogen using a pestle and mortar. Approximately 100 mg of ground tissue was used for RNA extraction. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RNA samples were treated with a DNAse treatment using the DNA-free™ DNA removal Kit (Thermo Fisher Scientific, Waltham, MA, USA). RNA quantity and quality were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Scientific™, Waltham, MA, USA) and visualised on a 2% agarose gel. RNA integrity was verified using an Agilent 2100 Bioanalyzer (Thermo Scientific™). In total, 22 samples were used for library preparation and sequencing (6 for experiment 1 and 16 for experiment 2).
Library preparation and Illumina high-throughput sequencing was performed by the NovoGene Sequencing company (Hong Kong, China) using the HiSeq paired-end platform (Illumina, San Diego, CA, USA). Sequencing depth was at least 50 million paired-end reads per sample. Sequence quality and error rate was verified using Q_phred scores.

To identify the genome editing events generated by mir-125b targeting gRNA and Cas9 mRNA-loaded RBCEVs, DNA were extracted from the EV treated MOLM13 cells using Trizol followed by phenol-chloroform and ethanol precipitation. The primary hsa-mir-125b-2 sequence (383 nucleotides) was amplified using a pair of gene-specific primers (sequences provided below) and Q5^® Hot Start high-fidelity master mix (New England Biolabs) in 30 cycles: 98 °C 10 s, 64 °C 30 s, 72 °C 45 s, and a final extension of 72 °C for 2 min. The PCR product was reamplified using the same primers with sequencing tags. High-through sequencing was performed by NovoGene Sequencing company (Hong Kong) using the HiSeq paired-end platform (Illumina, USA).