Truseq small rna library kit

Tumors were harvested from tumor-bearing mice after four weeks of ¹⁸⁸Re-liposome treatment. Total RNA of both saline control and ¹⁸⁸Re-liposome treated group were extracted using the QIAGEN RNA mini kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to manufacturer’s instructions. Furthermore, the quality of RNA was detected using the Nanodrop spectrophotometer (Nanodrop Technologies LLC, Wilmington, DE, USA). The integrity and concentration of RNA samples were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) with RNA 6000 nano kit (Agilent Technologies, Santa Clara, CA, USA). The TruSeq Small RNA Library kit (Illumina, Inc., San Diego, CA, USA) was then used to ligate RNA with adapters followed by the reverse transcription-PCR to generate cDNA library. The library was then sequenced by the HiSeq 4000 Sequencing System (2 × 150 bp paired-end Sequencing) and the results were processed with the Illumina software (Illumina, Inc., San Diego, CA, USA). To qualify the results of small RNA sequencing, the sequences were applied to the CLC Genomics Workbench v10 to obtain the qualified reads [91 ]. CLC Genomics Workbench counts different types of small RNAs in the data and compares them to databases of microRNAs or other small RNAs [92 (link)].

Small RNA libraries were made with the Illumina TruSeq Small RNA Library kit (Illumina, Inc., San Diego, CA), as per the manufacturer’s protocol. The ninety small RNA Illumina libraries were sequenced on a HiSeq 2500 (Illumina, Inc.) at the Iowa State University DNA Facility in Ames, IA. Reads were quality assessed using the FastQC program version 0.11.3 [113 ]. Reads were quality filtered and adapters were trimmed using Trimmomatic version 0.33 [114 (link)]. Reads were compared with the Rfam database using the Infernal program version 1.1.2 [115 (link)] and used to filter tRNAs, rRNAs, snoRNAs and snRNAs from the data. The reads were also filtered using the Triticeae Repeat Sequence Database [116 (link)] to remove any known Triticeae-specific repeat sequences. Two programs were used to identify sRNA candidates of interest from barley: miRDeep-P (version 1.3) and ShortStack (version 2.1.0) [33 (link), 34 (link)].

cDNA libraries were prepared by treating 1–5 μg total RNA or RNA extracted after HRDE-1 immunoprecipitation with 20 Units RNA 5′ polyphosphatase (Epicentre) in a total volume of 20 μl. De-phophorylated RNA was purified by phenol-chloroform extraction and ethanol precipitation according to standard protocols. Subsequent library preparations were performed with the TruSeq Small RNA library kit (Illumina) following the manufacturer’s instructions with exception that 15 cycles of PCR amplification were used. We size-selected cDNA libraries using 6% TBE PAGE gels (Life Technologies) and ethidium bromide staining. Desired sizes of cDNA bands were cut from the gel (between 147 and 157 nt), the gel matrix broken by centrifugation through gel breaker tubes (IST Engineering Inc.), and cDNA eluted with 400 μl of 0.3M Na-Chloride. Further purification of cDNA was by centrifugation through Spin-X 0.22μm cellulose acetate filter columns (Costar) followed by ethanol precipitation. Libraries were sequenced on a MiSeq Benchtop Sequencer or a HiSeq 2500 Sequencer (Illumina).

Messenger RNA (mRNA) sequencing libraries were prepared using the SMARTer Universal Low Input RNA kit, and sequenced (Illumina HiSeq 2000) with a paired-end 90 nucleotide read metric. Small RNA sequencing libraries were prepared using the TruSeq small RNA library kit (Illumina), and sequencing was conducted on the Illumina HiSeq 2000 platform. Raw sequencing data were trimmed of sequencing adaptors and low-quality reads removed using the “Trim Galore” package – a wrapper that incorporates CutAdapt and FastQC. For whole transcriptome analysis, quality-controlled reads were aligned to the Human Genome build hg19 using TopHat, a splice-junction aware mapping utility necessary for the successful mapping of intron-spanning (multi-exon) transcripts. Transcriptome assembly was performed using CuffLinks and a merged transcript representation of all samples produced using CuffMerge. Transcripts expressed at significantly different levels between the asthma and control samples were identified using CuffDiff, with a Q value ≤ 0.05 considered significant^{43 (link)}. MicroRNA (miRNA) analysis was performed by mapping miRNA reads to miRbase Version 21 using sRNAtoolbox^{44 (link)}. Differential expression of the miRNA reads was determined following statistical analysis with edgeR for R^{45 (link)}.

Small RNA libraries were generated from the same RNA samples that were used for matched transcriptomes. A total of six small RNA libraries were prepared for each strain, from 1 μg of total RNA each, using the TruSeq Small RNA library Kit (Illumina) following manufacturer’s instructions. 50 bp single-end libraries were sequenced on the HiSeq2500 instrument. Fastq files were assessed for quality using the FastQC (v. 0.11.3) package [27 ]. Adapters, low quality bases, and reads shorter than 13 bp were removed using the Cutadapt package (v. 1.8) [40 (link)]. Sequence reads without adapters were also discarded. Reads that passed QC were mapped to the genome sequence of S. carpocapsae, and microRNAs were identified by miRDeep2 (v. 2.0.0.8) [41 (link)], using a training set of mature and precursor microRNA sequences downloaded from miRBase (http://www.mirbase.org/). Naming of microRNAs was preferentially aligned with C. elegans, as indicated by miRDeep2 output. Novel S. carpocapsae microRNAs were named and numbered sequentially, taking care to avoid overlap with any C. elegans microRNA. Differentially expressed microRNAs were identified as above, using the DESeq2 package, and were presented using RColorBrewer, gplots, geneplotter and pheatmap packages.

Total RNA was extracted from the leaves of tomato plants with TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions, including a final treatment step with DNAse (Invitrogen Life Technologies), and the samples were purified with the GeneJet RNA Cleanup and Concentration Micro Kit (Qiagen, Hilden, Germany). Quality control of the total RNA profile was evaluated with the Qubit Fluorometer (Invitrogen Life Technologies) and the RNA 6000 Nano Kit as the 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA, USA). All samples were also run in a 1% agarose gel for visual quality evaluation. A total of 4 µg of RNA per sample were sent at a concentration of 200 ng/µL to ADM Lifesequencing (Valencia, Spain) for library construction and sequencing. Sequencing libraries were prepared by Illumina protocol based on the TruSeq Small RNA library kit, and, subsequently, Illumina sequencing-by-synthesis technology was used to generate the libraries, yielding over 185 million 75 bp single-end raw reads overall and a median of ca. 27.8 million reads per sample (Table 1).

sRNA libraries were prepared from total RNA as described previously (40 (link)). Briefly, 5 μg of DNase-treated total RNA was subjected to rRNA removal (RiboZero rRNA removal for Gram-positive bacteria; Illumina). rRNA-depleted samples were then subjected to RNA fragmentation using the Ambion RNA fragmentation kit (AM8740). Fragmented RNA was subjected to RNA 5′-polyphosphatase (Epicenter) treatment, which was performed to facilitate the 5′-adapter ligation step. Small RNA libraries were generated by Macrogen using the TruSeq small RNA library kit (Illumina). Then, 100-bp paired-end read sequencing was performed using an Illumina HiSeq2000 sequencer.