Quant it rna assay

RNA was collected from 3 separate differentiations including a combination of two WT clones and two SORL1KO clones. Each sample includes 2–3 technical replicates. RNA was collected from 2 million purified neurons for each sample. Purification of total RNA was completed using the PureLink RNA Mini Kit (Thermo Fisher 12183018A). Assessment of purified RNA was completed using a NanoDrop. Final RNA quantification was completed using the Quant-iT RNA assay (Invitrogen) and RNA integrity analysis was completed using a fragment analyzer (Advanced Analytical).

RNA was extracted from frozen brain tissues and iPSC-derived neuronal cell pellets using Tissue Lyser LT and RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA yields of individual samples were quantified by the Quant-iT RNA Assay (Life Technologies) on the Qubit Fluorometer (Fisher Scientific). Quality of yielded RNA of individual samples was assessed by the DV200 metric. DV200 of individual samples was measured with the RNA 6000 Pico Assay using the Bioanalyzer 2100 (Agilent Technologies). cDNA libraries of individual samples were constructed using the TruSeq Stranded Total RNA Sample Prep with Ribo-Zero Gold kit (Illumina) and then sequenced by the HiSeq 4000 (Illumina) at the McDonnell Genome Institute at Washington University in St. Louis with a mean of 58.14 ± 8.62 million reads as previously described^{23 (link)}.

Total RNA from flow-sorted cells was isolated by TRIzol-chloroform extraction. RNA samples were resuspended in Ambion Nuclease-free water (Life Technologies), snap frozen, and stored at -80 °C. Prior to RNA sequencing, RNA was treated with TURBO DNA-free kit (Invitrogen | Thermo Fisher Scientific, Waltham, Massachusetts, USA) and assessed using the Agilent Eukaryotic Total RNA 6000 and Quant-iT™ RNA assay kit on a Qubit™ Fluorometer (Life Technologies). cDNA was synthesized using the Ovation® RNA-Seq method, and the Illumina paired-end LT indexing protocol used to construct an Illumina library from 500 ng cDNA [19 (link), 30 (link)]. Libraries were sequenced on an Illumina HiSeq, and15-22Mbp per lane of 100 basepair paired-end reads generated. RNA-Seq paired-end reads were processed using the TopHat suite [44 (link)] with Cufflinks [36 (link), 37 ]. A fold-change and significance (< 0.05 False Discovery Rate, FDR) for every gene was generated using cuffdiff [43 (link)].