Truseq small rna library preparation kit

RNA was extracted from mycelium grown on putrescine toxin-induction medium using the same protocol outlined above for qRT-PCR, but without DNase I treatment. RNA samples were analyzed for integrity using either an Advanced Analytical Fragment Analyzer or an Agilent Bioanalyzer. RNA samples with RQN (RIN) scores >9 were accepted for sequencing. The 15–30 nt sRNA fraction was gel extracted to exclude genomic DNA and ribosomal RNA from sequencing as part of the Illumina TruSeq protocol. cDNA libraries of the sRNA fraction were prepared using the Illumina TruSeq Small RNA Library Preparation Kit (Illumina, San Diego, CA) with strict adherence to the manual. Briefly, the 3′ and 5′ RNA adapters were ligated on to fragments before a reverse transcription reaction was performed to synthesize cDNA. The cDNA library was further amplified with a PCR reaction. Next generation sequencing was performed on a MiSeq (Illumina) across multiple (2 or more) flow cells so that conditions and concentrations could be adjusted, if necessary, to achieve adequate read-depth. This is necessary due to the difficulty of adequately estimating the concentrations of sRNA preparations. All data files were uploaded to NCBI Bioprojects Sequence Read Archive at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA446058

Seventeen indexed miRNA libraries were prepared using the Illumina Truseq Small RNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA). Procedure for the preparation of the sequencing libraries were performed according to the manufacturer's protocol. Briefly, a 3′-adaptor sequence was first added to the 3′-end of the small RNA molecule then a 5′ adaptor sequence was added to the 5′-end of the small RNA. The adaptor ligated RNA was then reverse transcribed into cDNA, which was eliminated by adding RNase. The libraries were separated from adapter dimers by size fractionation in 8% TBE polyacrylamide gel (Life Technologies, Carlsbad, CA, USA). The 150 bp small RNA libraries were excised and purified from the gel. We then used the Agilent High Sensitivity DNA Kit (Agilent Technologies, Colorado Springs, CO, USA) to quantify the molarity and confirm the size distribution. The 17 Indexed miRNA libraries were pooled in equimolar concentrations and sequenced on one lane of an Illumina HiSeq 2000 platform (Illumina Inc., San Diego, CA, USA). The fastq files were produced using the CASAVA pipeline v2.0 (Illumina Inc., San Diego, CA, USA) and all generated sequence were deposited in NCBI dbGAP (accession number: phs001282.v2).

Small RNAs (sRNAs) were extracted from Jurkat cells using a mirVana miRNA Isolation Kit (Ambion) according to the manufacturer’s protocols. An additional selection of the entire sRNA fraction between 15–30 nt was performed by isolation from 15% denaturing PAGE and subsequent ethanol precipitation. sRNA sequencing libraries were prepared using Illumina TruSeq small RNA library preparation kit (Illumina) following the manufacturer’s instructions. In brief, 0.3-0.5 μg of sRNA fraction was ligated sequentially at the 3′ and 5′ end with synthetic RNA adapter, reverse transcribed and enriched in PCR with Illumina sequencing primers with barcodes. All libraries before the enrichment PCR were normalized by using qPCR with SYBR Green and with primers identical to Illumina TruSeq PCR primers but without end modifications. The number of cycles of subsequent enrichment PCR was determined based on the threshold cycle and was one cycle less than the threshold cycle. The amplified libraries were subsequently purified by 10% PAGE according to the expected product size. To validate the library efficiency and quantification, qPCR was carried out with SYBR Green Assays according to Illumina qPCR Library quantification protocol (Illumina). Normalized small RNA libraries were sequenced for 36 cycles, of three in a separate lane, using Illumina GAIIx Genome Analyzer (Illumina).