title: GSM4566699: PAS-seq_U1AMO_293; Homo sapiens; OTHER
library_construction_protocol: Total RNA from each sample was extracted by Trizol (Invitrogen) according to instructions. 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 containing index. Around 200 bp of PCR products was gel-purified and submitted for sequencing (single read 100 nucleotides).
attributes.tag: GEO Accession
attributes.value: GSM4566699