GSM1479377: mir-58 rep 3 [miRNA-Seq]; Caenorhabditis elegans; miRNA-Seq
RNA was isolated by TRIZOL (Life Technologies) according to the manufacturer's protocol and additionally purified by acidic phenol: chloroform (5:1) solution pH 4.5 (Ambion) / chloroform extraction to obtain high-grade RNA for sequencing. RNA concentration was measured by Nanodrop device (Thermo Fisher Scientific). 1 µg of total RNA isolated as described above was processed for small RNA sequencing. Four calibrator oligonucleotides (Cal 01-04 5 fMol each) were added as a reference as described previously (Hafner et al. 2012a; Hafner et al. 2012b). Briefly, RNA was dephosphorylated using FastAP (Fermentas) and radiolabelled according to the described method (Hafner et al. 2012a) with the 10 fold reduction of γ-32P-ATP (final concentration 0.05 μCi/μl). Subsequently, RNA was separated with denaturing PAA (15%) gelelectrophoresis and fractions between 18 and 25 nt in size were excised from the gel. Thereafter, preadenylated 3'-adapter (IDT DNA Technologies, 5'-TGGAATTCTCGGGTGCCAAGG-3') was ligated with truncated T4 RNA Ligase 2 (1-249, K227Q, final concentration 2000 U/µL, NEB) overnight on ice. After a denaturing 15% PAA gel separation, RNA ranging from 35 to 70 nt was cut out of the gel and extracted with 0.4 M NaCl overnight. The RNA was recovered by acidic phenol/chloroform extraction, ethanol precipitation, and addition of Glycoblue (Ambion) followed by 5'-RNA Adapter (5'-GUUCAGAGUUCUACAGUCCGACGAUC-3') ligation using T4 RNA ligase (Fermentas) at 37°C for 1 h. After an additional gel-purification step performed RNA was reverse transcribed into cDNA with SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's description. Library amplification step was performed with minimal number of PCR as determined by a preceding pilot PCR using different PCR cycle numbers. PCR was performed using Taq DNA-Polymerase (Sigma-Aldrich as described) with Index primers described in IlluminaTruSeq Small RNA Sample Prep Kit protocol. Amplicons were extracted using QiaExII (Qiagen) according to the purchaser's protocol. The recovered DNA was sequenced using single end, 50 cycle sequencing on HiSeq2000 (Illumina).
GEO Accession
GSM1479377