Total RNA was extracted from HEK293 cells with Qiagen RNAeasy kit according to the manufacturer's instruction. 2 µg were fragmented in a total volume of 100 µl fragmentation solution (10mM ZnOAc, 100mM Tris buffer pH 8.0) in a thin-walled PCR tube at 95 °C for 55 s.  Next, the samples were immediately cooled on ice and RNA was purified with RNA Clean and Concentrator kit (Zymo Research) according to the manufacturer's instruction. RNA was then treated with 10 U of calf intestine alkaline phosphatase in 1x CutSmart buffer (both from New England Biolabs) for 1h at 37 °C. Next, the samples were immediately cooled on ice and RNA was purified with RNA Clean and Concentrator kit (Zymo Research) according to the manufacturer's instruction. RNA was then treated with 20 U of T4 polynucleotide kinase in 1x T4 PNK reaction buffer (both from New England Biolabs) supplemented with 0.1 % (v/v) Triton-X100 for 30 min at 37 °C. Next, the samples were immediately cooled on ice and RNA was purified with RNA Clean and Concentrator kit (Zymo Research) according to the manufacturer's instruction. The next step was 3' adaptor ligation, which was performed for 16 h at 16 °C in a total volume of 20 µl in the presence of 2 U of T4 RNA ligase 2 truncated K227Q, 1x RNA ligase buffer, 25 % PEG-8000 (all from New England Biolabs) and 25 pmol of pre-adenylated 4N-RA3 adapter (see Oligonucleotides). Afterwards, RNA was separated on 10% Novex TBE Urea gels, and fragments in the range of 65-100 nt were excised and eluted in 300 mM NaCl overnight at 4 °C with 900 rpm agitation. RNA was then precipitated by the addition of 0.5 µl GlycoBlue co-precipitant, 3 volumes of absolute ethanol and incubation at -80 °C for at least 2 h. Next, precipitated RNA was reverse transcribed for 30 min at 37 °C in a total volume of 20 µl containing 1x buffer (25 mM Tris, pH 8.6, 30 mM NaCl and 5 mM dithiotreitol), 4.5 pmol RTP primer (see Oligonucleotides), 4 mM each dNTP, 20 mM MgCl2, 7.1 % (v/v) dimethylsulfoxide and 10 U of AMV reverse transcriptase (Promega). Afterwards, RNA was removed by RNase H (5 U) treatment for 30 min at 37 °C (New England Biolabs) and ssDNA was separated on 10% Novex TBE Urea gels. Fragments in the range of 65-100 nt were excised and eluted in 300 mM NaCl overnight at 4 °C with 900 rpm agitation. ssDNA was then precipitated by the addition of 0.5 µl GlycoBlue co-precipitant, 3 volumes of absolute ethanol and incubation at -80 °C for at least 2 h. The next step was 5'adaptor ligation, which was performed for 16 h at 22 °C in a total volume of 20 µl. The reaction contained precipitated ssDNA, 60 U of high concentration T4 RNA ligase 1, 1x RNA ligase buffer, 22.5 % PEG-8000, 1 mM ATP (all from New England Biolabs) and 50 pmol of 4N-SRC-cDNA adapter (see Oligonucleotides). ssDNA was purified with DNA Clean and Concentrator kit (Zymo Research) according to the manufacturer's ssDNA protocol. PCR amplification for the final library was performed according to the Truseq small RNA protocol (Illumina) using standard indexed PCR primers (RPIx) for multiplexing (Illumina). Typically, 14 cycles of amplification were required to obtain a sufficient amount of the final cDNA library in the size range of 150-220 bp (average size 180 bp). PCR reactions were separated on a 2.5 % agarose gel and the final DNA library of the expected size range was excised.
GEO Accession
GSM4458919