Truseq small rna sample prep kit

We extracted total RNA from 30 pairs of ovaries and testes in 2–4-day-old adults, according to the manufacturer’s instructions (Macherey-Nagel). PolyA mRNAs were extracted using the “FastTrack MAG Micro mRNA isolation kit” (Life Technologies), fragmented with RNA fragmentation reagents (Ambion), and treated with antarctic phosphatase (NEB) and polynucleotide kinase (NEB), according to the recommendations. We prepared strand-orientated libraries with the “Truseq Small RNA sample prep Kit” (Illumina). The final gel purification step has been replaced by a polymerase chain reaction cleanup with AMPureXP beads (Beckman-Coulter). We then proceeded to mRNA libraries illumina sequencing.
Small RNAs were extracted from total RNA of 50 pairs of ovaries and 100 pairs of testes, dissected from 2-to 4-day-old adults, using a TRIzol extraction according to the manufacturer’s procedure (TRIzol reagent, Invitrogen). We size fractionated small RNAs from 1 µg total RNA on a TBE-urea 15% acrylamide gel. We treated the resulting RNAs with the Illumina “Truseq Small RNA sample prep Kit” according to the manufacturer’s recommendations and send small RNA libraries to deep sequencing.

A total of 6 samples (isolated from the NRCMs) were used for total RNA extraction using the TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. The quality of the RNA samples was examined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and standard denaturing agarose gel electrophoresis. Small RNA library preparation was performed using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA). The quality-ensured RNA-seq libraries were then sequenced using Illumina Hiseq2000/2500. The identification of known miRNAs (mapped to the miRbase database) and read counting were processed using ACGT101-miR (LC Sciences, Houston, TX, USA). A modified normalization was used to correct copy numbers among different samples, and a miRNA was considered present when the normalized read count was >0 in all the samples. A heatmap was constructed using the normalized read counts of the known miRNAs in each EV sample using R (R version 4.0.3) with a heatmap via a custom written R script [46 (link)].

Exosome miRNA sequencing was performed on 16 plasma samples (10 patients with STEMI and 6 healthy volunteers) for the test group. The small RNA sequencing library was prepared using the TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, California, United States). RNA integrity was then examined through the BioAnalyzer 2100 (Agilent, California, United States). Approximately, 100 ng of RNA were used to prepare a small RNA library according to the protocol of the TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, California, United States). Then, the single-end sequencing (1 bp × 50 bp) was performed on Illumina HiSeq 2500 by LC-Bio (Hangzhou, China) following the vendor’s recommended protocol. The raw data of high-throughput sequencing was deposited at the GSE 185729 in the National Center for Biotechnology Information (NCBI).

The NanoDrop-2000 spectrophotometer was supplied by NanoDrop Technologies (Wilmington, DE, USA). The TRIzol reagent was supplied by Invitrogen (Life Technologies, Carlsbad, CA, USA). The TapeStation 2200 (Agilent Technologies Inc., Santa Clara, CA, USA), Illumina TruSeq™ Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA), Qubit® 2.0 Fluorometer using the dsDNA HS and/or BR assay kit (Life Technologies, Carlsbad, CA, USA), Illumina HiSeq 2000 sequencer (Illumina, San Diego, CA, USA), 36–50 kDa DSS (#160110, MP Biomedicals, CA, USA), Bio-Rad CFX96TM Real-Time PCR system (Bio-Rad Laboratories, CA, USA), TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA), RNase inhibitor (Thermo Fisher Scientific, Rockford, IL, USA), and iQTM SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) were obtained from the indicated manufacturers.

Total RNA extraction was performed using TRIzol reagent (Invitrogen, MA). Six pairs of degenerative NP samples (100 mg each) and matched controls were tested. An Agilent 2100 Bioanalyzer (Agilent, CA) was utilized to examine RNA integrity (RIN). The library construction was performed using an Illumina TruSeq small RNA Sample Prep kit (Illumina, CA) and 1 μg of total RNA following the manufacturer’s instructions. Fifteen cycles of PCR were carried out. The quality of the RNA product was examined with a High Sensitivity DNA Chip and an Agilent 2100 Bioanalyzer (Agilent Technologies, CA), and the concentration was assessed by qPCR using a KAPA Library Quantification kit (KAPA Biosystems, CA). Each library was adjusted to a concentration of 20 pM and was sequenced with a MiSeq Reagent kit v3 for 150 cycles by an Illumina MiSeq Sequencing System (Illumina). An efficiency of 25 million sequence reads per flow cell was achieved.

Isolation of total RNA, including small RNA, was performed with the miRNeasy kit (#217004; Qiagen, Hilden, Germany) according to the manufacturer's protocol with the following alterations: 1 mL of Qiazol reagent was mixed with 0.2 mL of serum, the entire aqueous phase was loaded onto a single column from the MinElute Cleanup Kit (#74204; Qiagen), and RNA was eluted in 20 μL of RNase‐free water. One‐fourth (5 μL) of the RNA isolated from each serum sample was used to construct sequencing libraries with the Illumina TruSeq Small RNA Sample Prep Kit (#RS‐200‐0012; Illumina, San Diego, CA, USA), following the manufacturer's protocol. Briefly, 3′ and 5′ adapters were sequentially ligated to small RNA molecules and the obtained ligation products were subjected to a reverse transcription reaction to create single stranded cDNA. To selectively enrich fragments with adapter molecules on both ends, the cDNA was amplified with 15 PCR cycles using a common primer and a primer containing an index tag to allow sample multiplexing. The amplified cDNA constructs were gel purified and validated by checking the size, purity, and concentration of the amplicons on the Agilent Bioanalyzer High Sensitivity DNA chip (#5067‐4626; Genomics Agilent, Santa Clara, CA, USA). The libraries were pooled in equimolar amounts and sequenced on an Illumina HiSeq 2000 instrument to generate 50‐base reads.