Qiaseq mirna library construction kit

Purified, lyophilized peptide-modified preE_M-let-7f was resuspended in LC-MS grade water and quantified via optical density measurement at 260 nm. The QiaSeq miRNA library construction kit (Qiagen, Cat#331502) was used to generate a library from ∼100ng of peptide-modified RNA using the standard protocol, incorporating 16 cycles of PCR. For quality assurance, Tape Station High Sensitivity tape is run on the Tape Station 2200 from Agilent. Additionally, a qPCR run was completed using the Kapa Library Quantification Kit (Kapa, KK4824) with a standard 0 for normalization. Generated libraries were pooled together in equimolar proportions and were denatured per standard Illumina protocol. Sequencing was performed on an Illumina MiSeq with Nano protocol at 6 pM with 50% PhiX for a read length of PE 75 and depth of up to 1 million reads.

Trizol reagent (1 mL) was added to the exosome suspension, and RNA was extracted following the Trizol reagent manual. The RNA was precipitated in 1:1 isopropanol (v/v) and 1 µL glycogen at −20 °C overnight. An exosomal miRNA library was constructed with a QIAseq miRNA library construction kit (QIAGEN) used in accordance with the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq-2500 sequencer for 50 cycles. Reads that passed the Illumina quality filters were retained for analysis. Adapters were trimmed from the reads and reads shorter than 17 nt were discarded. The remaining reads were then mapped to the miRBase human miRNA reference database using the FANSe3 algorithm on the Chi-Cloud NGS Analysis Platform (Chi-Biotech Co. Ltd., Shenzhen, China) (21 (link)).