title: Illumina HiSeq 2500 paired end sequencing; The transcription of long non-coding RNAs is regulated by EGF in MCF10A cell line
design: The transcription of long non-coding RNAs is regulated by EGF in MCF10A cell line
library_construction_protocol: MCF10A cells (1.5 x 10E6) were seeded onto 60 mm dish in Complete Medium without EGF and with 0.5 % Horse serum instead of 5 %. After 48 hrs incubation, EGF was added to a final concentration of 20 ng/ml for the EGF treated group. After 30 minutes, nuclear fractions were isolated according to the protocol described previously (PMID: 22955620). Briefly, cells were washed with 1xPBS and then incubated for 10 min with 1 ml of RLN buffer (50 mM TrisHCl pH=8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% (v/v) IGEPAL CA-630 and 2 U/ml superaseIn – RNase inhibitor, Invitrogen AM2696). Subsequently, the lysate was centrifuged at 1 000 g for 5 min at 4°C, the pellet was collected, resuspended in 1 ml of RLN buffer, incubated for a further 5 min on ice and centrifuged at 1 000 g for 5 min at 4°C. At this stage nuclear fractions were stored at -80°C for subsequent RNA extraction. The RNA was extracted from the nuclear fraction using an RNeasy plus kit (Qiagen, 74134) with DNase treatment according to the manufacturer’s protocol. cDNA libraries from two biological replicates were prepared following the TruSeq Stranded mRNA sample preparation protocol (Illumina)