Truseq small rna kit

To resolve the transcript sequences more precisely, RNA was sequenced through Illumina (Megji) sequencing. Total RNA was extracted from carp skeletal muscle tissue samples, and Nanodrop2000 was used to measure the concentration and purity of the proposed RNA. RNA integrity and integrity number were determined by agarose gel electrophoresis and Agilent 2100, respectively. OligodT enrichment was subsequently performed to isolate the mRNA. One-strand cDNA was synthesized by reversing the mRNA template, followed by double-strand synthesis to form a stable double-stranded structure linking the adaptors. After library enrichment and purification of the products (6% Novex TBE PAGE gel; 1.0 mm, 10 wells) and sequencing of the short sequence fragments using the Illumina Novaseq 6000 platform, eukaryotic libraries were constructed using the Illumina TruSeq Small RNA kit. Small RNA statistics require the sequencing of small (18–32 nt) enriched RNA fragments; therefore, the Illumina TruSeq Small RNA kit was used to build the eukaryote library.

For RNA isolation, exosomes were prepared using PEG precipitation, resuspended in cold PBS and centrifuged in Optima XPN-100 Ultracentrifuge (Beckman Coulter) at 100,000 x g, 4°C for 75 min. After ultracentrifugation, pellets were resuspended in 200 μl of ice cold Exosome Resuspension Buffer (Total Exosome RNA & Protein Isolation Kit, Invitrogen) and stored at -80°C. RNA was isolated using the manufacturer’s protocol. For preparation of a library of RNAs longer than 200 nucleotides, TruSeq Stranded mRNA kit (Illumina) was used. Library of miRNAs was prepared using the TruSeq small RNA kit (Illumina). Both libraries were sequenced on the Illumina NextSeq 500 machine. Single-end reads of 75 bp were sequenced for the long RNAs and 50 bp single-end reads—for the small RNAs.

RNAs were pretreated to modify 5’ ends in two ways. For RPPH treatment, 1-2 μg of total RNA was treated with 10 units (2 μl) RNA 5’ pyrophosphohydrolase (NEB) for 1 h at 37°C. The treated RNA was phenol-chloroform extracted and ethanol precipitated with sodium acetate and glycogen for 2 days, and resuspended in RNase-free water. For CIP-RPPH treatment, 3-5 μg of total RNA was treated with 4 μl of QuickCIP (Quick dephosphorylation kit, NEB) in a total volume of 40 μl for 90-120 min at 37°C. RNA was phenol-chloroform extracted, precipitated overnight with sodium acetate and glycogen and resuspended in RNase-free water.
Small RNA libraries from treated or untreated RNA were built using the TruSeq small RNA kit (Illumina) according to the manufacturer’s instructions except for an increase in the number of PCR cycles from 11 to 15. Libraries were eluted in 0.3 M NaCl, ethanol precipitated and quantitated with Qubit and TapeStation. Libraries were pooled in groups of 6 to 12 per lane and sequenced on an Illumina HiSeq2000.
The Illumina universal adapter was trimmed from small RNA reads using cutadapt v1.10 and reads were mapped to the corresponding genome assemblies with Bowtie v0.12 (Langmead et al., 2009 (link)) with parameters –v 0 –m 1.

Five micrograms of total RNA was pretreated with either 20 U of TAP (Epicentre) or 20 U of RNA 5′ polyphosphatase (Epicentre) in a volume of 20 µL for 45 min at 37°C or used directly. After standard extraction, RNA was used for TruSeq small RNA library preparation according to the manufacturer's instructions (Illumina), including 15 cycles of PCR. Libraries were sequenced on a MiSeq instrument (Illumina). A detailed description of processing and analysis of the data thus generated are in the Supplemental Material.
Purification of nuclear short RNAs was performed as described (Chen et al. 2013 (link)). Briefly, nuclei of 2 million young adults grown in liquid culture were isolated as described in Ooi et al. (2010) (link), and RNA was extracted using Tripure (Roche). Short capRNA-seq libraries were cloned from 20 µg of nuclear RNA as follows: After size selection for RNAs of 20–100 nt, we performed RNA polyphosphatase (Epibio) treatment followed by Terminator exonuclease (Epibio) treatment and 3′ adapter ligation. After treatment with heat labile alkaline phosphatase (Epibio), capped RNAs were rendered accessible for cloning by TAP treatment. 5′-adapter cloning and library generation were completed as described in the TruSeq small RNA kit (Illumina), and sequencing was performed on a HiSeq instrument (Illumina, SE50).

Total RNA was extracted from longissimus dorsi muscle of Ningxiang pigs by Trizol reagent (Invitrogen, Life Technologies, Carlsbad, CA, United States) following the manufacturer’s procedure. The amount and purity of the extracted RNA were detected by Nanodrop 2000 (NanoDrop Technologies, Wilmington, DE, United States), RNA integrity was detected by agarose gel electrophoresis and RIN value was determined by Agilent 2100 (Agilent Technologies, Santa Clara, CA, United States). A single library construction requires a total RNA of 5 μg, concentration ≥250 ng/μL, OD260/280 between 1.8 and 2.2. Ribosomal RNA was removed using the Ribo-Zero Magnetic kit (Epicentre, Madison, WI, United States). Next, TruSeqTM Stranded Total RNA Library Prep Kit is used to construct a strand specific library to detect circRNA, lncRNA and mRNA. A small RNA library was constructed using Illumina TruSeq Small RNA kit to detect miRNA. The strand specific library and small RNA library were sequenced on HiSeq 4000 platform by PE150 and SE50 respectively. Deep sequencing was performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China).