Total RNA was isolated using the hot phenol method as described in (Lybecker et al., 2014). total DNaseI treated RNA was depleted of ribosomal RNA using the Ribo-Zero™ RNA removal kit for Gram-negative bacteria (Epicentre). A 3' RNA adapter, based on the Illumina multiplexing adapter sequence (Oligonucleotide sequences © 2007-2014 Illumina, Inc. All rights reserved) blocked at the 3' end with an inverted dT (5'-GAUCGGAAGAGCACACGUCU[idT]-3'), was phosphorylated at the 5' end using T4 PNK (New England Biolabs) per the manufacturer’s protocol. The 3' RNA adapter was ligated to the 3' ends of the rRNA depleted RNA using T4 RNA ligase I (New England Biolabs). 1.5 mg of RNA was incubated at 20°C for 6 hours in 1X T4 RNA ligase reaction buffer with 1 mM ATP, 30 µM 3' RNA adapter, 10 % DMSO, 10 U of T4 RNA ligase I, and 40 U of RNasin (Promega) in a 20 ml reaction. RNA was then fragmented in equivalents of 100 ng using the RNA fragmentation reagents (Ambion®) per the manufacturer’s protocol at 70°C for 3 min and subsequently phosphorylated at the 5' ends using T4 PNK (New England Biolabs) per the manufacturer’s protocol to allow for ligation of the 5' adapter. RNA was size-selected (≈ 150-300 nt) and purified over a denaturing 8 % polyacrylamide/8 M urea/TBE gel. Gel slices were incubated in RNA elution buffer (10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.1 % SDS, 0.3 M NaOAc) with vigorous shaking at 4°C overnight. The supernatant was subsequently ethanol precipitated using glycogen as a carrier molecule. The Illumina small RNA 5' adapter (5'-GUUCAGAGUUCUACAGUCCGACGAUC-3') was ligated to the RNA as described before except the concentration of the adapter was 52 mM and 20 U of T4 RNA ligase I was used in total volume of 25 µl. The ligated RNAs were size-selected (≈ 200-300 nt) and gel-purified over a denaturing 8 % polyacrylamide/8 M urea/TBE gel (as described above). The di-tagged RNA libraries were reverse-transcribed with SuperScript®II reverse transcriptase (Invitrogen) using random nonamers per the manufacturer’s protocol. RNA was removed using RNase H (Promega) per the manufacturer’s protocol and cDNA was amplified in PCR carried out using Phusion® High-Fidelity Polymerase (New England Biolabs). cDNA was amplified with modified designed Illumina-compatible PCR primers (3’ library Forward 5’-CAAGCAGAAGACGGCATACGACAGGTTCAGAGTTCTACAGTCCGA-3’; Reverse 5’-AATGATACGGCGACCACCGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3’) by 18 cycles of PCR. The products were purified using Agencourt AMPure XP beads (Beckman) and analyzed on an Agilent 2100 Bioanalyzer. 3’ end enriched cDNA libraries were sequenced on individual Genome Analyzer IIx lanes (36 bp, single-end) or on HiSeq 2000 lanes (50 bp, single-end) using primer based on Illumina Multiplexing Read 2 Sequencing Primer (5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3’) at the CSF NGS unit http://csf.ac.at/.
GEO Accession
GSM2608040