nucleic acid sequencing protocol
Multiplexed samples were sequenced with the IlluminaHighSeq system. Each library was sequenced as 76-mers (paired end).
nucleic acid library construction protocol
4 ug RNA extracted from biological duplicate samples was poly (A) enriched by incubating with oligo(dT) coated sepherose beads for two rounds (Ambion). The resultant enriched mRNA was fragmented, size selected and reverse transcribed according to the IlluminaTruSeq RNA Sample Preparation Guide.
nucleic acid extraction protocol
Trizol (Invitrogen) was used for extraction of total RNA from hESCs treated with control shRNA or SON shRNA for 3 days.
For transfection, the hESCs were passaged with 0.25% trypsin (Invitrogen) for dissociation and transfected with 1.5 ug shRNA and 4.5 ulMirus LT1 transfection reagents the following day. Cells were harvested for mRNA 3 days after transfection of SON shRNA1.
The hESC line H1 (WA-01, passage 30) was cultured feeder-free in conditioned medium on Matrigel (BD). Conditioned medium contains 20% Knock-out serum replacement (Gibco), 1 mM L-glutamine (Gibco), 1% non-essential amino acids (Gibco) and 0.1 mM 2-mercaptoethanol (Gibco), 4 ng/ml human basic fibroblast growth factor (bFGF) (Invitrogen), and medium was supplemented with additional bFGF (8ng/ml) before use. The hESCs were passaged for expansion with 1 mg/ml collagenase IV (Gibco) every 5-7 days.