Truseq rapid sbs kit hs

Total RNA was extracted from the yeast cells using TRIzol LS Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The concentration of RNA was calculated using the Quantus fluorimeter and QuantiFluor RNA System kit (Promega, Fitchburg, WI, USA). The quality control was performed with QIAxcel Advanced System (Qiagen, Hilden, Germany) capillary gel electrophoresis. The lower threshold for RIS quality control of the samples was no less than 5. The RNA libraries were prepared with the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, USA), NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs), and oligonucleotide indexing set NEBNext Multiplex Oligos for Illumina, Index Primers Set 1 (New England BioLabs) from 1 µg of the total RNA.
The whole transcriptome RNA sequencing (RNA-Seq) was performed with the HiSeq 2500 sequencing platform (Illumina, San Diego, CA, USA) in the paired-end mode and with a read length of 2 × 100 bp using the TruSeq Rapid PE Cluster Kit—HS (Illumina) and TruSeq Rapid SBS Kit—HS (200 cycles) (Illumina).

In preparation for next generation sequencing (NGS) a total of 72 RNA samples from n = 6 animals for MC and SM samples, respectively were prepared in a library preparation using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England BioLabs, MA, USA) described by our group previously [29 (link)]. In brief, adaptor sequences were ligated to 200 ng total RNA starting material. The RNA was reverse transcribed, amplified by PCR and barcoded for 24x multiplexing. The DNA concentrations of all samples were determined using a DNA 1000 chip on a Bioanalyzer 2100 (Agilent Technologies, CA, USA). The samples were pooled and fractions were size-selected via high resolution gel electrophoresis. The gel bands were cut at 135–151 bp, corresponding to small RNA size of 16–32 bp. The DNA libraries were controlled regarding correct size, purity and concentration with a High Sensitivity DNA chip on a Bioanalyzer 2100. Single-read sequencing-by-synthesis was carried out in 50 cycles on a HiSeq 2500 platform (Illumina, CA, USA) using the TruSeq^® Rapid SBS Kit—HS (50 cycle) and TruSeq^® Rapid SR Cluster Kit—HS (Illumina). Each flow cell was loaded with each 12 MC and 12 SM samples to assure reproducible conditions.