RNA sequencing in human iPSCs derived from bipolar patients to identify important therapeutic molecular targets of Valproate(VPA)

Valproate(VPA) has been used in the treatment of bipolar disorder since the 1990s. However, the therapeutic targetsof VPA have remained elusive. Here we used RNA sequencing in human iPSCs derived from bipolar patients to further identify important molecular targets. Human iPSCs were homogenized and total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany). RNA quantity and quality were assessed using fluorometry (Qubit RNA Broad Range Assay Kit and Fluorometer; Invitrogen, Carlsbad, CA) and chromatography (Bioanalyzer and RNA 6000 Nano Kit; Agilent, Santa Clara, CA), respectively. Libraries were prepared using TruSeq Stranded mRNA (PolyA+) kit (Illumina, San Diego, CA) and sequenced by Illumina NextSeq 500. The read length was 75bp with 30-40M reads per sample. FastQC (v0.11.3) was performed to assess data quality. TopHat2 (v2.0.9) aligned the reads to the mouse reference genome (Mus musculus UCSC mm10) and to the Ensembl human reference genome (GRCh38.p13) using default parameters. Alignments were then converted to expression count data using HTseq (v0.6.1) with default union mode. Human fibroblasts or lymphoblasts from multiple patients were reprogrammed to hiPSCs via non-integrating episomal-mediated, lentivirus-mediated, or retrovirus mediated gene transfer, characterized, and differentiated to NPCs, as previously described (Supplementary Table 2). We used iPSCs from two patients diagnosed with bipolar disorder and classified as Lithium Non-Responsive (Pt-LiNR-1 and Pt-LiNR-2). Patient selection criteria, clinical evaluation and classification were conducted as thoroughly described previously. We use two separate clones from each patient in these experiments. Differentiation into neurons was similar to techniques described previously (PMID: 28500272). Neurons were cultured for 4 weeks prior to pharmacological experiments. Neurons were cultured in four 12-well plates of cells with two plates as technical replicates for each patient. Each of the wells of the 12-well plate were treated with either vehicle (water), VPA (1mM), or ACY957 (3μM), repeated in quadruplicates (2 patients x 2 clones x 3 treatments x 4 replicates = 48 samples). Cells were treated for 72 hrs with daily media and compound changes, then collected, washed (1X PBS), and pelleted then flash frozen for RNA extraction.