GSM3721132: rst1-3_rep3; Arabidopsis thaliana; ncRNA-Seq
RNA was extracted with TRI-reagent (MRC) following the manufacturers instructions. After precipitation with 0.8 Vol isopropanol for 1-3 h at -80 °C, RNAs were collected by centrifugation (30 min 16000 x g, 4 °C), washed twice with 70% EtOH, resupended in water and further purified by adding 1 Vol of Phenol:Chloroform:Isoaylalkohol (25:24:1). The aqueaous phase was transferred in a fresh tube, RNAs were precipitated over night with 2.5 Vol of EtOH at -80 °C, collected by centrifugation, washed twice with 70 % EtOH and resupended in water to approx. 2,5 µg/µl. Libraries were prepared from 1 µg of total RNA using the NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB #E7300S and #E7580S) following the manufacturer's instructions. After ligation of primers, cDNA synthesis and PCR amplification, pooled libraries were loaded on a NOVEX 6% polyacrylamide gel with 100 mM Tris, 100 mM Borate, 2 mM EDTA. Five fractions corresponding to 130-180 nt products were excised and eluted over night in water. After EtOH precipitation, size and concentration of the fractions were checked with an Agilent 2100 Bioanalyzer (Agilent Technologies). The fraction of 140-150 nt containing the 21-22 nt small RNAs of interest was sequenced on a HiSeq 4000 sequencer (single-end mode 1x50bp).
GEO Accession
GSM3721132