RNA-seq of MCF10A transduced with an inducible lentiviral MYB expression system
RNA-seq of MCF10A transduced with an inducible lentiviral MYB expression system
Cells were detached by addition of trypsin. Cells suspensions were quenched with medium and centrifuged at 400 x g for 5 minutes to pellet for subsequent extraction. Cells were cultured in DMEM/F12 with 5% horse serum and 100 U/ml penicillin/streptomycin, supplemented with 20 ng/ml human epithelial growth factor (HeGF), 500 ng/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 ug/ml insulin. Doxycycline was added to the culturing medium at 2 ug/ml. Total RNA was extracted using Monarch Total RNA Miniprep Kit according to manufacturer's instructions (New England Biolabs, Hitchin, UK). Library construction was performed by Novogene UK (Cambridge, UK). A minimum of 1 ug (20 ng/Î¼l) of high-quality total RNA (using Monarch Total RNA Miniprep Kit according to manufacturer's instructions, New England Biolabs, Hitchin, UK) was supplied for sequencing. Novogene UK constructed cDNA libraries by purification of messenger RNA using poly-A enrichment. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification.