Truseq sbs kit v3 hs chemistry

The libraries were diluted according to a cluster generation protocol and loaded
into a v3 Illumina Flowcell (16 samples per line with four technical replicates;
the technical replicates were used to avoid the lane effect). Single-read
clusters were generated on a cBot system (Illumina). Flowcell clustering was
performed using TruSeq SR Cluster Kit v3-cBot- 4 HS. Sequencing-by-synthesis of
the clustered libraries was conducted on a HiScanSQ System in 81 bp single-end
cycles using TruSeq SBS Kit v3-HS chemistry (Illumina).

In total, 42 blood samples were collected from the jugular vein into Tempus™ Blood RNA Tubes (Ambion, Life Technologies) and stored at −20 °C. Total RNA was isolated using the MagMAX™-96 Total RNA Isolation Kit (Ambion, Life Technologies) according to the manufacturer’s protocol. The quality and quantity of obtained RNA were examined using NanoDrop 2000 (Thermo Scientific; Wilmington, USA) and TapeStation 2200 instruments with Agilent RNA ScreenTape (Agilent, Perlan Technologies). The RNA samples with RIN (RNA integrity number) values above 8.5 were used for further analysis. Five samples were removed from RNA-seq analysis due to the exclusion of horses from the training cycle for various reasons (lameness or poor metabolic performance).
The cDNA libraries were prepared from 400 ng of total RNA with TruSeq RNA Sample Prep Kit v2 kit (Illumina) according to the protocol. The quality and quantity of the obtained libraries were established using Qubit 2.0 (Invitrogen, Life Technologies) and TapeStation 2200 (D1000 ScreenTape; Agilent). RNA sequencing was performed with a 75-bp single-end run on a HiScanSQ platform (Illumina) using TruSeq SR Cluster Kit v3- CBOT-HS and TruSeq SBS Kit v 3 - HS chemistry (Illumina). Indexed samples were pooled into two groups and loaded into 3–4 lanes on two flow-cells, which allowed for obtaining at least 3 technical replications per library.