Truseq rapid sbs kit

For primary blood PMN samples involving cell isolation, we used a pooling strategy involving equal mixing of total RNA samples derived from independent biological samples. Pooling of small RNA samples is effective in reducing data variability, and it reduces the number of replicates, hence lowering the cost for subsequent steps [64 (link)]. For each of the other samples, a unique total RNA specimen was analyzed.
The quality and the concentration of the total RNA samples were verified using a NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA) and gel separation (Supplementary Table S15). The sRNA-Seq libraries, containing RNA species between 8 and 30 nt in length were prepared as previously described [65 (link)]. Samples were diluted to a final concentration of 8 pM, denatured as single-stranded DNA, and cluster generation was performed on the Illumina cBot using a TruSeq Rapid SR cluster kit (GD-402-4001, Illumina, San Diego, CA, USA). Afterward, the clusters were sequenced for 51 cycles on Illumina HiSeq 2000 using TruSeq Rapid SBS Kits (FC-402-4002, Illumina, San Diego, CA, USA), as per the manufacturer’s instructions.

Total RNA from PRV Fa ΔgE/gI strain-infected, Fa wild-type strain-infected, and non-infected PK-15 cells was ligated to 3′ and 5′ adapters with T4 RNA ligase. cDNA was synthesized and amplified using RT primers and amplification primers (Illumina, San Diego, CA, USA). PCR-amplified products of 120–140 bp were purified, and the complete libraries were tested by the Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA samples were adjusted to 8 pM, then cluster generation was sequentially performed on the Illumina cBot system (Illumina). High-throughput sequencing was performed on an Illumina HiSeq 2000 using TruSeq Rapid SBS Kits (Illumina), according to the manufacturer’s instructions.