Truseq small rna sample preparation kit v2

Small RNA (18~30 nt) libraries for SW480/SW620 cells and derived EVs (sMVs and exosomes) were constructed using Illumina TruSeq Small RNA Sample Preparation Kit v2 and sequenced on a HiSeq 2000 platform (Illumina), according to the manufacturer’s protocols. Briefly, small RNAs (18~30 nt) were fractioned on a 15% Tris-borate-EDTA (TBE) polyacrylamide gel (Life Technologies, CA) from total RNA (200 ng), purified by centrifugation, and ligated with adaptors. Small RNAs were then reverse transcribed into cDNAs and amplified using the adaptor primers for 14 cycles. The cDNA fragments (~150 bp) were isolated from a 6% TBE PAGE-gel and directly used for cluster generation by using TruSeq PE Cluster Kit v3 (Illumina). Using TruSeq SBS Kit v3 (Illumina) biological replicates (n = 2) were sequenced for each sample.

Total RNA (8 μg) of each sample was processed to remove the ribosomal RNA using the Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA). Then, the rRNA-depleted RNA sample was used for the library construction using the NEBNext^® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, USA), as described (Zhang et al., 2018 (link)). Small RNA sequencing libraries of the two rabbit breeds were constructed using the Illumina TruSeq Small RNA Sample Preparation Kit v2, as described (Chen et al., 2019b (link)). Both stranded transcriptome sequencing and small RNA sequencing were performed on the HiSeq 2500 platform in NovoGene (Beijing, China).