title: Illumina HiSeq 2500 paired end sequencing; RNA-Seq of human embryonic stem cells over expressing NANOS3 and DAZL
design: RNA-Seq of human embryonic stem cells over expressing NANOS3 and DAZL
library_construction_protocol: The hESC line HS401 (46,XY) was cultured on hESC-qualified Matrigel (Corning) -coated plates using mTeSR1 medium (StemCell Technologies) in 5% CO2 and atmospheric oxygen at 37°C. The cells were passaged using Accutase (Life Technologies), followed by an over night incubation with 5 μM Y-27632 (Millipore). hESCs were co-transfected with 2.5 μg piggyBac transposon (IFP2) and 2.5 μg transposase vector (MOCK, NANOS3, or DAZL) in a 6-well format using 5 μl PLUS reagent and 10 μl Lipofectamine LTX (Life Technologies) according to the manufacturer’s instructions. Two days after the transfection, cells were selected with 1 μg/ml puromycin (Life Technologies) for 6 days. Transfection was repeted three times for each transposase vector and cells were collected for RNA isolation at passage 1 after transfection. RNA libraries for sequencing were prepared using TruSeq Stranded mRNA Sample prep kit with 96 dual indexes (Illumina, CA, USA) according to the manufacturers instructions with the following changes. The protocols were automated using an Agilent NGS workstation (Agilent, CA, USA) using purification steps as described in: S. Lundin/ H. Stranneheim PLoS ONE 2010) and (E. Borgstrom/ S.Lundin et al. 2011 PLoS ONE).