Nextflex small rna seq kit v3

Small RNA sequencing (sRNA-Seq) was performed on RNA isolated from blood plasma from Senegalese sole treated (n = 16) and non-treated with kisspeptin (n = 15) (see details in Table 3). SncRNAs were isolated from blood plasma samples using miRNeasy Serum/Plasma Kit (Qiagen, Germany) following the manufacturer’s instructions, and assessment of RNA quality and quantity was performed on a 2200 TapeStation Nucleic Acid system using High Sensitivity RNA ScreenTapes (Agilent, Santa Clara, CA, USA). Libraries were prepared from 30 samples (Table 3) using NEXTflex Small RNA-Seq kit v3 (Bio Scientific, Phoenix, USA) for Illumina platforms following the manufacturer’s protocol. Library size, purity, and concentration were evaluated on a High Sensitivity D1000 ScreenTape (Agilent, USA). Normalized libraries were pooled at equimolar concentrations and multiplexed sequencing was done in two independent runs on a NextSeq500 sequencer using a NextSeq High Output kit v2 (75 cycles; Illumina, San Diego, CA, USA). All sequencing data were submitted to the NCBI SRA database under the accession number GSE153469.

miRNA-seq libraries were constructed using either the NEXTflex Small RNA-Seq Kit v3 (Bio Scientific, Austin, TX) for sample IDs 003 to 032, or QIAseq miRNA library kit (Qiagen) for sample IDs 033 to 107, following the manufacturers’ protocols, respectively. Briefly, 3’ and 5’ adapters were added to small RNAs present in the plasma sample in this order. Reverse transcription was performed to convert the target small miRNAs into cDNAs. An assigned index was given to each sample for multiplexing. A 22-cycle of PCR amplification was performed. After size selection by magnetic beads, DNA fragments with the correct insert sizes were selected for the miRNA-seq library. Libraries were quantified using Qubit 3.0 HS dsDNA assay (Thermal Fisher Scientific, Waltham, MA). Library size and quality were examined using the TapeStation 2200 (Agilent, Santa Clara, CA). The miRNA-seq libraries were sequenced, 76 bp, single-end, on an Illumina NextSeq 550 (Illumina, San Diego, CA) with a final loading concentration of 2.1 pM.

Small RNA sequencing libraries were prepared using NEXTFLEX Small RNA-Seq Kit v3 (Bio Scientific Corp., TX, United States) using the manufacturer’s protocol. Briefly, 500 ng of total RNA was used as starting material. 3′ adapters were ligated to the specific 3′OH group of microRNAs followed by ligation of 5′ adapter. Adapter-ligated fragments were reverse transcribed with Moloney murine leukemia virus (M-MuLV) reverse transcriptase by priming with reverse transcription primers. Complementary DNA (cDNA) thus formed was enriched and indexed by PCR (15 cycles). Libraries were size selected using the NEXTflex magnetic bead purification system and finally reconstituted in nuclease-free water. The sequencing libraries were quantified by the Qubit fluorometer (Thermo Fisher Scientific, United States), and its fragment size distribution was analyzed on Tape Station using Agilent D1000 Screen Tapes (Agilent Technologies, United States). The libraries were sequenced on Illumina NextSeq 500 sequencer (Illumina, Inc., United States) for 75-bp single read chemistry following manufacture’s guidelines.