Qiabeads magnetic beads

The preparation of the miRNA library was done using the QIAseq miRNA Library Kit (Qiagen, Germany). For this, a total of 100 ng (from the granulosa cells) and 50 ng (from EVs) of total RNA were converted into miRNA NGS library. After ligating adapters with unique molecular identifiers (UMI) to the 5′ and 3′ ends of the RNAs, cDNA was synthesized using reverse transcription (RT) and during the RT reactions, unique PCR indices were added. Following that a clean-up of the cDNA was performed using the Qiabeads magnetic beads (Qiagen, Germany), and the cDNA samples were amplified (16 cycles) using the indexing forward and universal reverse primers. Following the library amplification, a clean-up of the library was performed with Qiabeads magnetic beads (Qiagen, Germany). Library quality control was performed using Bioanalyzer 2100 (Agilent). Based on the quality of the insert and concentration, libraries were pooled in equimolar ratios and pooled libraries were quantified using qPCR. Pooled libraries were sequenced on a NextSeq550 sequencing platform (Illumina, CA) according to the manufacturer instructions. On average, 14 million Single-end reads were generated per samples with a read length of 75 nt. Raw data files were de-multiplexed and the FASTQ file of each sample was generated using the bcl2fastq software (Illumina, CA).