Multiplex small rna library prep set

HEK293T cells in a 6-well format were transfected with 1-ug of an individual cshRNA vector. Forty-eight hours post-transfection, small RNAs (<70 nts) were isolated from total RNA as described in (20 (link)) and pooled. Small RNA libraries were prepared for Illumina small RNA sequencing from the pooled small RNAs using the multiplex small RNA library prep set (New England Bio Labs) and sequenced on a Illumina HiSeq 2500 and NextSeq 500 platforms (GSAF, UT Austin). Adapter sequences were trimmed from the reads using fastx clipper from the FASTX-Toolkit software (http://hannonlab.cshl.edu/fastx_toolkit). The preprocessed reads were then mapped to cshRNA reference sequences using SHRiMP2 software package (23 (link)). Small RNA reads were quantitated using custom scripts.

piRNA sequencing was performed by Novegene (Beijing, China). The small RNAs (sRNAs) of serum exosomes from 4 HCC patients and 4 non-tumour donors were randomly enrolled for piRNA sequencing by using the Multiplex Small RNA Library Prep Set for Illumina^® (San Diego, CA, USA) according to the manufacturer’s instructions. After quantification and quality control (Agilent 2100 pic600, Santa Clara, CA, USA), the sRNAs were added to 3′ and 5′ adapters, and a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed. The products were gel-purified and length filtered according to the range of length. The sRNA tags were matched to genome version GRCh38.
Then, we mapped the reads (mismatch=1) with piRNABank (http://pirnabank.ibab.ac.in/)19 (link) to obtain the known piRNA profiles in serum exosomes. The unmapped reads were used to predict the novel piRNAs according to a k-mer scheme designed by Zhang et al.20 (link) Then, we obtained the reads of 4 bases in each position of the piRNAs. The base distribution of piRNAs is shown. The differentially expressed serum exosome-derived piRNAs between HCC patients and non-tumour donors were screened by using the DESeq R package at the threshold of log₂ fold change > 1 and adjusted p value < 0.05.