Illumina HiSeq 2000 paired end sequencing; RNA-Seq profiling of dopaminergic neurons from iPSCs of Parkinson's disease patients
RNA-Seq profiling of dopaminergic neurons from iPSCs of Parkinson's disease patients
Total RNAs were extracted with Trizol reagent (Invitrogen) and purified with RNeasy Mini kit (Qiagen), according to the manufacturers’ instructions.  NPCs were generated using an adapted protocol (Li et al., 2011). NPCs were regularly cultured in maintenance contained N2B27 medium supplemented with 3 μM CHIR99021, 2 μM SB431542, 5 μg/mL BSA, 20 ng/ml bFGF and 20 ng/ml EGF. Differentiation of NPCs into mDA neurons was initiated one day after passaging the NPCs on Poly-L-ornithine hydrochloride/Laminin coated culture. NPC maintenance medium was substituted by mDA patterning medium consisting of N2B27 medium supplemented with FGF8, Purmorphamine, Dibutyryl cAMP (db-cAMP) and L-ascorbic acid (L-AA) for 14 days. From days 14, cells were fed with maturation medium consisting of GDNF, BDNF, DAPT, db-cAMP and L-AA. unspecified Total RNAs were extracted with Trizol reagent (Invitrogen) and purified with RNeasy Mini kit (Qiagen), according to the manufacturers’ instructions. Ribosomal-RNAs were depleted from 4μg total RNAs by using Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) (Epicentre Illumina). And RNA sequencing libraries were constructed by using 20ng ribosomal RNA-depleted total RNA samples with NEBNext® UltraTM RNA Library Prep Kit for Illumina® (New England Biolabs). RNA sequencing at a 51bp single-end read scale was performed on an Illumina Platform HiSeq2000 (Genome Institute of Singapore, Singapore), using ribosomal RNA-depleted RNA from two biological replicates of the wild type cell lines GM23280A, GM23279A and IR1.7 and PD iPSC lines ND34391, ND35367, ND38477 cell at NPC stage and mDA stage.   For detecting alternative splicing events, RNA sequencing libraries were generated from intact poly (A)+ RNAs prepared by using Oligo d(T)25 Magnetic Beads (New England Biolabs). RNA sequencing was conducted on an Illumina Platform HiSeq2000 at a 101bp paired-end read scale (Genome Institute of Singapore, Singapore)
Experimental Factor: Healthy
disease