GSM5914559: BT474 cells-A1_EtOH; Homo sapiens; RNA-Seq
Total RNA was extracted from the cell lysates using the RNeasy mini kit (QIAGEN) according to the manufacturer's instructions. RNA library preparation was performed following the NEB NEBNext Ultra Directional Library Preparation protocol. Briefly, 350 ng of total RNA was used as the input material. The material was enriched for poly-A mRNA, fragmented into the 200-300-bases range for 4 minutes at 94oC and converted to double stranded cDNA, end-repaired and adenylated at the 3' to create an overhang A to allow for ligation of Illumina adapters with an overhang T; library fragments were amplified under the following conditions: initial denaturation at 98oC for 10 seconds, followed by 12 cycles of: 98oC for 10 seconds, 60oC for 30 seconds and 72oC for 30 seconds, and finally an extension step for 5 minutes at 72oC; at the amplification step, each sample were amplified with a different barcoded adapters to allow for multiplex sequencing. One ul of the final RNA libraries was loaded on a Bioanalyzer 2100 DNA High Sensitivity chip (Agilent Technologies) to check for size; RNA libraries were quantified by qPCR using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems).