3.0 fluorometer

For the RNA-seq of EECs, total RNA was prepared using an RNeasy Plus Micro Kit (Qiagen, Dusseldorf, Germany). The RNA purity was assessed using a Nano Drop spectrophotometer, the RNA concentration was assessed using a Qubit 3.0 fluorometer and the RNA integrity was assessed using an Agilent 2100 Bioanalyzer. The sequencing library was prepared following the instruction manual of a VAHTS total RNA-seq (H/M/R) Library Prep Kit for Illumina^® (Vazyme, China). To isolate the appropriate cDNA fragment size for sequencing, the library fragments were selected with VAHTSTM DNA Clean Beads. The enzyme UDG was used to digest the second strand of cDNA. PCR amplification was performed, and the products were purified. After cluster generation, the libraries were sequenced on an Illumina Hiseq X10 platform (Illumina, California, USA), and 150-bp paired-end reads were generated. Raw reads in the FASTQ format were first processed using in-house Perl scripts. Then, the processed reads were mapped to the reference genome. The reference genome and gene model annotation files were downloaded directly from the genome website. The reference genome index was built using hisat2-build^{14 (link)}, and paired-end clean reads were aligned to the reference genome using Hisat2 (v2.0.5)¹⁵ .

Mouse MII oocytes were obtained as described above. A single MII oocyte was transferred to a PCR tube with lysis buffer containing dNTP and oligo (dT) oligonucleotides. Then, a sequencing library from a single oocyte was constructed using a SMART-seq HT Kit (Takara). Purified libraries were quantified and validated by a Qubit 3.0 Fluorometer and Agilent 2100 Bioanalyzer to confirm the insert size and calculate the concentration. Then, sequencing was performed on an Illumina NovaSeq 6000 (Illumina). RNA-seq reads were aligned to the GRCm38.100 reference genome using Hisat2 software. For subsequent downstream analysis, the original expression reads were converted to FPKMs according to the method described above.

The RNA-seq of monocytes was performed via the Smart-Seq2 method. Briefly, samples were collected in tubes with lysis components and ribonuclease inhibitors. An Oligo-dT primer was introduced to the reverse transcription reaction for first-strand cDNA synthesis, followed by PCR amplification to enrich cDNA and a MagBeads purification step to clean up the product. Then, the cDNA product was checked with a Qubit^® 3.0 Fluorometer and Agilent 2100 Bioanalyzer to verify the expected product with a length of approximately 1~2 kb. Next, the cDNA was randomly sheared by ultrasonication according to the Illumina library preparation protocol, which included DNA fragmentation, end repair, 3’ end A-tailing, adapter ligation, PCR amplification and library validation. After library preparation, the PerkinElmer Lab ChIP^® GX Touch system and the Step OnePlus™ Real-Time PCR system were used for library quality inspection. Qualified libraries were then loaded on the Illumina HiSeq platform for PE150 sequencing. Sequencing data were mapped to the human reference genome (ucsc.hg19) using HISAT2 v2.1.0. fragments per kilobase of transcript per million mapped reads (FPKM) values for each gene were calculated. RNA-seq data were shown in Supplementary Data Sheet 1 and the quality control of RNA-seq was in Figure S1.

To prepare poly(A)-enriched RNA sequencing libraries, we processed 500 ng of total RNA using the TruSeq Stranded mRNA Library Prep kit (Illumina, Cat # 20020595; Indexes: IDT for Illumina TruSeq RNA UD Indexes, Cat # 20020591). The resulting libraries were then analyzed on a TapeStation (Agilent) and quantified via a Qubit 3.0 fluorometer. Equimolar amounts of the resulting sequencing libraries were combined to make a 4 nM pool. The pools were then sequenced on a Novaseq 6000 instrument (Illumina) in single-end 100 bp format at the Genomics Core at the Institute for Genomic Medicine at UCSD or the La Jolla Institute for Immunology.

RNA concentration and integrity were obtained using Qubit 3.0 fluorometer and Agilent 2100 Bioanalyzer, respectively. Briefly, 10 ng of total RNA was reverse transcribed and the resulting cDNA was amplified for 12 cycles (cell samples) or 16 cycles (EVs samples) by adding PCR Master Mix and the AmpliSeq mouse transcriptome gene expression primer pool (targeting 20 767 well-annotated RefSeq genes + 3 163 XM and XR genes, based on UCSC mm10). Amplicons were digested with the proprietary FuPa enzyme and ligated onto barcoded adapters. The library amplicons were bound to magnetic beads. Libraries were amplified, re-purified and individually quantified using Agilent TapeStation High Sensitivity tape. Individual libraries were diluted to a final concentration of 80 pM and pooled equally for each group of samples for further processing. Emulsion PCR, templating and 540 chip loading was performed with an Ion Chef Instrument (Thermo Fisher Scientific). Sequencing was performed on an Ion S5XL™ sequencer (Thermo Fisher Scientific).

Total RNA was subjected to library preparation using the NEBNext Ultra II directional RNA library prep kit for Illumina (New England Biolabs, Beverly, MA, USA), according to the manufacturer’s instructions. Size selection was used to select for fragments ranging between 150–200 bp in length. RNA libraries were quantified using a Qubit^® 3.0 fluorometer as described above and their fragment size range was assessed by high-resolution, chip-based gel electrophoresis with a Bioanalyzer 2100 instrument (Agilent Technologies, Waldbronn, Germany) and the Agilent DNA7500 Kit (Agilent Technologies, Waldbronn, Germany). Libraries were pooled equimolarly and sequenced in paired-end (2 × 150 bp) mode on a HiSeq 2000 instrument (Illumina, Munich, Germany). Transcriptome sequencing (RNA-seq) was performed by Eurofins Genomics (Constance, Germany).