title: GSM7077608: Diff_D1_Protocol_P1_Line_DF6-9-9T.B_FUS_P525L_homozygous_Cell_1203; Homo sapiens; RNA-Seq
library_construction_protocol: Neuronal cultures were treated for 1 hour with 5 µM Y-27632 before dissociation with trypsin or accutase. The cells were resuspended in FACS buffer (HBSS with magnesium and calcium, 2% w/v bovine serum albumine, 2% v/v knockout serum replacement, 2% v/v B-27 supplement, 25 mM D-glucose, 5 µM Y-27632). Single live cells were isolated by fluorescence activated cell sorting (FACS) using Topro-3 iodide and were directly lysed in ice-cold single-cell lysis buffer (0.2% w/v Triton X-100, 0.5% v/v recombinant RNase inhibitor, 10 µM Smarter_oligo-dTVN, 25 mM dNTP, 100 mM DL-dithiothreitol, 4e-5 diluted ERCC spike-in RNAs, in ultrapure water). Protocol according to Schweingruber C, et al. 2023. Methods Mol Biol (PMID: 36264494). Individual single cell libraries were prepared using the Smart-Seq2 protocol followed by tagmentation using components form the Nextera XT DNA library preparation kit (Illumina). Protocols with adaptations according to Schweingruber C, et al. 2023. Methods Mol Biol (PMID: 36264494).