Agarose

Libraries for sequencing were prepared from 1.1 μg of total RNA obtained from rat samples (20 SE and 20 controls) and patients (16 mTLE and 8 controls). Libraries were prepared using a NEXTflex Small RNA-Seq Kit v2 for human samples and Kit v3 for rat samples (Bioo Scientific) according to the manufacturer’s protocol. After the amplification step, samples were analyzed using a Fragment Analyzer (Advanced Analytical), and specific fragments of the miRNA library (145 bp) were quantified. Samples were equally pooled for each particular sequencing run based on the concentration of miRNA library fragments, which were isolated using the Pippin Prep instrument using the 3% agarose (Sage Science) prior to sequencing. Isolated fragments were quantified using NanoDrop ND-1000 (Thermo Fisher Scientific) and Qubit^® 2.0 fluorometer (Thermo Fisher Scientific) and used for sequencing on NextSeq 500 (Illumina) according to the manufacturer’s protocol.

Libraries for massively parallel sequencing (MPS) were prepared from 10.5 µL of isolated miRNAs using Kit v3 (Bioo Scientific) according to the manufacturer’s protocol. Fragments after the amplification step were analyzed using a Fragment Analyzer (Advanced Analytical), and fragments of the miRNA library (145 bp) were quantified. Samples were pooled in equal concentrations of miRNA library fragments, which were isolated using the Pippin Prep system with 3% agarose (Sage Science) before sequencing. The isolated fragments were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and were used for sequencing on a NextSeq 500 (Illumina) according to the manufacturer’s protocol.

Sequencing libraries were prepared from 1.1 µg of total RNA obtained from 10 SE and 10 control animals per time point in both age groups (n = 120), using a NEXTflex Small RNA-Seq Kit v3 (#NOVA-5132–06, Bioo Scientific) according to the manufacturer’s protocol. After the amplification, samples were analyzed using Fragment Analyzer (Advanced Analytical), and specific fragments of the mature miRNA library (145 bp) were quantified. Samples were equally pooled based on the concentration of miRNA fragments, which were isolated by the Pippin Prep instrument using the 3% Agarose (Sage Science) prior to sequencing. Isolated fragments were quantified using Qubit™ dsDNA HS Assay Kit on Qubit® 2.0 Fluorometer (Thermo Fisher Scientific) and used for sequencing on NextSeq500 (Illumina) according to the manufacturer’s protocol.