title: GSM3556977: SB.DOX_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted by Qiagen miRNeasy Mini Kit (QIAGEN Inc.) according to the manufacturer's instructions. CAGE libraries were prepared as previously described (Murata et al., Methods Mol Biol 2014). Briefly, 3 μg of total RNA from each sample was subjected to reverse transcription using SuperScript III Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) with random primers. The 5-end cap structure were biotinylated by sequential oxidation with NaIO4 and biotinylation with biotin hydrazide (Vector Laboratories, Burlingame, CA, USA). After RNase I treatment (Promega, Madison, WI, USA), the biotinylated cap structure was captured with streptavidin-coated magnetic beads (Life Technologies). After ligation of 5' and 3' adaptors, second-strand cDNAs were synthesized with DeepVent (exo−) DNA polymerase (New England BioLabs, Ipswich, MA, USA). The double-stranded cDNAs were treated with exonuclease I (New England BioLabs) and purified.
attributes.tag: GEO Accession
attributes.value: GSM3556977