title: GSM1213445: HCT116_24h_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were rinsed once with 1xPBS, and lysed in 3ml of Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA extraction was carried out according to the manufacturer’s instructions. The concentration of purified total RNAs was adjusted to 1 μg/μl with RNase free water. Ten μg of total RNA was subjected to 15% TBE-urea PAGE before small RNA species between 18-30nt in length were excised and eluted in 0.3M NaCl. The v1.5 small RNA 3’ adapter (Illumina) was ligated using T4 RNA ligase 2, truncated (NEB), and then the small RNA 5’ adapter was ligated using T4 RNA ligase. Reverse transcription was performed using the small RNA RT primer (Illumina) using the 5’ and 3’ adapter-ligated RNAs as a template. Twelve cycles of PCR amplification were carried out using GX1 and GX2 primers with Phusion DNA polymerase. The amplified cDNA preparation was subjected to 6% TBE PAGE, and species of ~100bp were excised and purified. Finally, each cDNA library was validated with an Agilent bioanalyzer using the DNA 1000 kits to confirm size, purity, and concentrations.
attributes.tag: GEO Accession
attributes.value: GSM1213445