GSM8387722: WT, id 4, treated; Mus musculus; RNA-Seq
After a rapid wash in PBS (Sigma D8537) to remove cellular debris, total RNA was extracted fromprimary cortical neurons using PureZOL (BioRad 7326890). In details, 0.3 mL of PureZOL (12wells plate) was added on cell culture and left 5 min at room temperature to allow the completedissociation of nucleoprotein complexes. 200L of chloroform was then added for 1 ml of PureZOLaccording to the manufacturer's protocol. Following, samples were inverted for 15-20 seconds togently mix the phenol:chloroform mixture and incubated 3 min at RT. After centrifugation at12000g for 15 min at 4°C, the upper aqueous phase was transferred into RNAse free tube andprecipitated with 10 ug of RNA-grade glycogen and 100% isopropanol 1:2. Samples were storedo.n. at -20°C. The day after, the RNA was centrifuged at 12,000g for 10 min at 4°C and pellets werethen washed in 500ul of 70% ethanol (7,500 x g for 5 min at 4°C). To remove genomic DNA,sample pellets were treated with DNAse (Sigma-Aldrich, AMPD1) at 37°C for 15 min. A secondpurification with PureZOL was executed on RNAs and their pellets were finally resuspended in 10uL of RNAse-free H2O. Total RNA was extracted from eight WT and eight KO cortical neurons (14 DIV) treated with trehalose and eight WT and eight KO untreated controls as described above for a total of 32 samples. Only high-quality RNA with an RNA Integrity Number (RIN) of 8 or higher was used. Library preparation and sequencing have been performed with TruSeq RNA Library Preparation Kit v2 (Illumina) by Genewiz. One WT untreated and corresponding treated sample and two KO untreated and related treated samples were found outliers and removed from the dataset.