title: NextSeq 500 paired end sequencing; RNA-seq of TGF-beta and knockdown of histone variant H2A.Z induced EMT in MDCK cells.
design: RNA-seq of TGF-beta and knockdown of histone variant H2A.Z induced EMT in MDCK cells.
library_construction_protocol: MDCK cells were maintained in DMEM growth medium supplemented with 10% Foetal Bovine Serum (FBS) Cells for lentiviral transduction were grown to 70% confluency. A final concentration of 4μg/mL polybrene (SIGMA) and 10mM HEPES were combined with lentiviral-containing supernatent pre-warmed to 37 ̊C and added to cells. After incubation at 37 ̊C/5%CO2 for 24 hours, lentiviral-containing supernatant was removed and replaced with fresh complete medium. Cells were allowed to grow for another 24 hours, before harvest. Cells were washed and resuspended in 1% FBS/PBS. A 100% population of transduced cells were obtained by sterile cell sorting for GFP tagged cells.  Total RNA was extracted usign Qiagen RNAeasy kit following manufacturer's instructions. Libraries were constructed according to the Illumina TruSeq stranded mRNA protocol with poly-A enrichment.
identifiers.namespace: Research Fellow Tremethick Lab Department of Genome Sciences The John Curtin School of Medical Research The Australian National University
identifiers.id: E-MTAB-5628:MDCKshZD6_1_p