GSM6042962: Perk KO neurons, Control, Biol Rep. 2; Mus musculus; RNA-Seq
Cells were washed with ice-cold PBS and cell lysates were prepared by incubation and scraping in TRI Reagent   solution (Invitrogen). Cell lysates were loaded on Phase Lock Gel-Heavy tubes (Quantabio) and mixed 1:5 with chloroform (VWR Chemicals). The organic   and aqueous phase were separated by centrifugation at 12000 g for 15 min at 4°C. The aqueous phase was mixed 1:1 with Isopropanol (VWR Chemicals) and 150 mg/mL glycoblue (Invitrogen) and centrifuged at 12000 g for 10 min at 4°C to precipitate RNA. Precipitated RNA was washed with 75% ethanol (Sigma-Aldrich) and centrifuged 7500 g for 5 min. After removal of ethanol, the pellet was air-dried and dissolved in RNase-free H2O. Transcriptome analysis was performed by Novogene (UK) Company Limited, Cambridge, United Kingdom. Quantity and quality of isolated RNA was assessed by agarose gel electrophoresis, spectrophotometry using the NanoPhotometer (IMPLEN) and the RNA Nano 6000 assay kit of the Bioanalyzer 2100 system (Agilent). Only qualified samples were used for RNA sequencing. First, sequencing libraries were generated, using 1 µg RNA per sample and   the NEBNext Ultra TM RNA Library Prep Kit for Illumina (New England BioLabs), according to the manufacturer's protocol and index codes were added to attribute sequences. PCR products were purified using the AMPure XP system and library quality was assessed using the Bioanalyzer 2100 system (Agilent). Clustering of the index-coded samples was performed using a cBot Cluster Generation System and PE Cluster Kit cBot-HS (Illumina), according to the manufacturer's protocol. Finally, the library preparations were sequenced on a NovaSeq 6000 platform (Illumina) and paired-end reads were generated.