Ligation to a 5-adenylated 3-adapter (5-rApp/NNNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC) together with the truncated T4 RNA ligase II (NEB) and to a 5 adapter (5-GUUCAGAGUUCUA CAGUCCGACGAUC) by T4 RNA ligase I (NEB). The resultant RNA was reversetranscribed with Superscript III (Invitrogen), followed by 12 cycles of PCR amplification with Phusion high fidelity polymerase (NEB). cDNA libraries had been sequenced on an Illumina HiSeq 2500. Filtered poly(A) site-supporting (PASS) reads had been made use of to construct peaks applying the Cufflinks application. Obtained peaks have been linked with all the UCSC assembly of human genes determined by the peaks position applying the closest-features command of BEDOPS toolkit. PASS mapped within the 5000 nucleotide area downstream with the annotated gene were thought of as novel three UTR isoforms. This annotation was applied to exclude TRENDseq reads originating from internal priming on the genome encoded adenosine-rich regions. For mouse annotation of correct poly (A) web sites, data (PMID 24072873) were applied with settings identical for human neuroblastoma annotation. Nucleic acid high quality assurance. RNA integrity was assayed with Agilent RNA 6000 Nano Kit (Agilent Technologies) as outlined by the Curdlan medchemexpress manufacturer’s instructions74, and also a threshold of minimal RNA integrity number of 9.5 was applied for total RNA. Homogeneity and size of DNA libraries for Illumina sequencing have been analysed applying Agilent Higher Sensitivity DNA Kit (Agilent Technologies) following the manufacturer’s guidelines. Qubit?2.0 Fluorometer in combination with dsDNA HS Assay Kit (ThermoFisher Scientific) was utilised to assay cDNA library concentration. TRENDseq: library preparation and sequencing. Total RNA (100 ng) was reverse-transcribed in presence of oligonucleotide (RT) primer containing T7 promoter, Illumina five adapter, individual in-lane barcode and an anchored oligodT stretch, as described previously75. For the cDNA and aRNA synthesis MessageAmp II aRNA Amplification Kit (ThermoFisher Scientific) was utilised as outlined by the manufacturer’s recommendations with modifications. Especially, for the initial and second cDNA strand synthesis for every single individual RNA input sample 1/10 of full reaction size was utilised. Up to 25 samples had been pooled soon after second cDNA strand synthesis reaction. In vitro transcription (aRNA synthesis) was performed in 40 reaction format in accordance with the manufacturer’s protocol with 14 h of incubation at 37 . Purified aRNA was sheared working with Covaris M220 FocusedUltrasonicatorTM (Peak incident power 50 Watt, Duty Factor 20 and 200 Cycles per Burst (cbp) for 420 s at 7 ) and size-selected on the 6 Web page in denaturing conditions (7 M urea). The gel area corresponding to 100 nucleotides was excised, and RNA was eluted from gel by two min incubation in 50?00 of buffer containing 100 mM Tris-HCl (pH 8.0), 500 mM NaCl and 1 SDS at area temperature. Size-selected RNA was purified applying miRNeasy Kit (Qiagen). Illumina platform compatible cDNA library was synthesised as described previously75 using a number of PCR cycles reduced down to 9. Each pooled library (up to 25 samples) was labelled with Illumina indexing barcode and up to 3 libraries have been pooled collectively adding as much as 75 samples per sequencing run. The libraries were sequenced around the Illumina HiSeq or NextSeq platform with addition of 30 PhiX Sequencing control (Illumina) in the paired-end setup. Read 1 (9 nt) sequenced individual sample in-lane barcode (introduced within the first reverse transcription-step) and study 2 (50 nt).
