Small rna primer set

sRNA library preparation and sequencing were performed with Illumina sequencing technology. The sRNA library was generated with the TruSeq Small RNA Library Kit. Briefly, 3′ and 5′ adapters were ligated into 1 μg of total RNA with T4 RNA ligase. Then, reverse transcription was performed with the Illumina sRNA RT-Primer, and cDNA was amplified by PCR (11 cycles) using the Illumina small RNA primer set. The amplified total cDNA library was purified and size selected (insert size 22–30 bp) in 6% Novex TBE gel. The transcriptome libraries were sequenced on a HiSeq2500™ (Illumina) with the following parameters: SE50 (single end) and 10 M clean reads, which yielded a minimum of 500 Mb of raw data per sample (SE50, 10 M, 500 Mb). The sRNA-seq data were submitted to the NCBI Sequence Read Archive (SRA): SRP150595. The detailed analysis of the regulated miRNA has been published elsewhere [9 (link)]. In this study, we focus on multiple different classes of sRNAs that are differentially expressed in the blood in response to IA rupture. We reanalyzed profiles of miRNA regulation to compare and contrast with the other classes.

Four equal pools (5 individuals) of total RNA were obtained from the PL7, PL10, PL13 and PL16 prawns. The samples for microRNA transcriptome analysis were prepared using a Truseq^TM Small RNA Sample Prep Kit (Illumina, San Diego, USA). Total RNA was ligated with proprietary 5′ and 3′ adapter. Adapter-ligated small RNA was then reverse transcribed to create cDNA constructs using Superscript reverse transcriptase (Invitrogen, CA, USA). The cDNA constructs were subsequently amplied by 15 cycles of PCR using Illumina small RNA primer set and Phusion polymerase (New England Lab, USA), and purified on 6% Novex TBE polyacrylamide gel. Sequencing cycle number is 50 and the reads length is 50 nt with single end sequencing pattern.

Sequencing libraries were prepared according to the Solexa Small RNA Sample Prep Protocol. Briefly, the RNA was resolved on 15% polyacrylamide denaturing gels. Gel fragments with a size range of 18–40 nucleotides were excised, and the small RNA fragments were eluted overnight with 0.5 M NaCl at 4°C, and precipitated by ethanol. The small RNA was ligated to 5′ and 3′ RNA adapters with T4 RNA ligase, transcribed into cDNA by Super-Script II Reverse Transcriptase (Invitrogen) and amplified by 15 PCR cycles using Illumina™ small RNA primer set that were annealed to the ends of the adapters. The amplified cDNA products were purified and recovered. Finally, Solexa/Illumina HiSeq sequencing technology was employed to sequence the small RNA samples (BGI, Shenzhen, China). The sequencing data was deposited in the Gene Expression Omnibus (GEO accession GSE58941).