RNA-seq of SOX5 overexpressing primary human neuronal progenitors

Purpose: The goal of this study was to assess gene expression changes in neurons overexpressing SOX5 using human primary neuronal culture system. Methods: 6 samples each from control GFP and SOX5 overexpressing neurons were used to isolate total RNA using miRNeasy kit, Qiagen. We performed rRNA-depleted 69bp paired end stranded RNA-seq on neurons overexpressing either GFP or SOX5 tagged with GFP. Overexpression of SOX5 in neurons validated that a significant proportion of Attenuated cortical patterning (ACP) genes are regulated by SOX5, and that predicted SOX5 targets exhibit a net downregulation, consist with its repressive function. This supports the prediction that attenuated patterning of SOX5 between cortical regions contributes to direct alterations in SOX5 targets and likely to indirect alterations in SOX5 non-targets in the ACP set. delpleted 69 bp stranded RNA-seq in Overall design: SOX5 was overexpressed in primary human neuronal cultures using a lentiviral system. Briefly full-length human SOX5 gene was cloned in pLVU/GFP vector (gift from Lars Ittner [Addgene plasmid #24177]) using the gateway recombination technique. Lentivirus was produced in HEK293T cells using a second generation packaging vector system (psPAX2, a gift from Didier Trono [Addgene plasmid #12260] and pCMV-VSV-G, a gift from Bob Weinberg [Addgene plasmid #8454]) as described by Stewart et al., 200331. Primary human neurons were infected at plating at a multiplicity of infection (MOI) of 10 with either the SOX5 overexpressing construct or a control pLVU-GFP backbone vector. 14 days after infection, RNA from the samples were isolated using miRNeasy micro kit (Qiagen, Carlsbad) and 50bp paired-end libraries were prepared using SMARter Stranded Total RNA sample prep kit (Clontech) with rRNA depletion. Libraries were then multiplexed and sequenced with HiSeq 2500 instrument (Illumina).

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Parikshak NN, Swarup V, Belgard TG, Irimia M, Ramaswami G, Gandal MJ, Hartl C, Leppa V, Ubieta LT, Huang J, Lowe JK, Blencowe BJ, Horvath S, Geschwind DH