GSM2664294: MEF cells treated with Nudt21 siRNA and induced to express OKSM for 3 days _D3_replicate 2  [PAS-seq]; Mus musculus; OTHER
RNA isolation was performed using the miRNeasy mini kit (Qiagen). RNA was eluted from the columns using RNase-free water and quantified using a Nanodrop ND-1000. Total RNA was extracted with Trizol as per manual (Life technologies), 10 μg total RNA was fragmented with fragmentation reagent (Ambion) at 70 oC for 10 minutes followed by precipitation with ethanol. After centrifugation, RNA was dissolved and Reverse transcription was performed with PASSEQ7-2 RT oligo : [phos]NNNNAGATCGGAAGAGCGTCGTGTTCGGATCCATTAGGATCCGAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTT[V-Q] and Superscript III. cDNA was recovered by ethanol precipitation and centrifugation. 120-200 nucleotides of cDNA was gel-purified and eluted from 8% Urea-PAGE. Recovered cDNA was circularized with CircligaseTM II (Epicentre) at 60 oC overnight. Buffer E (Promega) was added in cDNA and heated at 95 oC for 2 minutes, and then cool to 37 oC slowly. Circularized cDNA was linearized by adding BamH I (Promega). cDNA was centrifugated after ethanol precipitation. PCR was carried out with primers PE1.0 and PE2.0 contaning index. Around 200 bp of PCR products was gel-purified and submitted for sequencing (single read 100 nucleotides).
GEO
GSM2664294
GEO Accession
GSM2664294