title: GSM8548413: A196V-A196L_patient_10_repl2; Homo sapiens; OTHER
library_name: GSM8548413
library_construction_protocol: Cells were lysed in LiDS/LET buffer (5% LiDS in 20 mM Tris, 100 mM LiCl, 2 mM EDTA, 5 mM DTT, pH7.4, 100 μg/ml Proteinase K). Lysates were incubated at 60°C for 10 minutes, pushed 10 times through a 1-ml syringe with a 26G needle, and mixed by vortexing. Two volumes of cold acid phenol (pH 4.3), 1/10 volume 1-Bromo-3-chloropropane and 50 µg glycogen were added. Samples were mixed vigorously by vortexing, followed by centrifuging at 10,000 g, 4°C. The aqueous phase was transferred to a new tube and the phenol/BCP extraction was repeated. RNA was precipitated by the addition of 3 volumes of 100% ethanol and incubation at -20°C for 30 min. Pellets were washed with 80% ethanol, air-dried, and resuspended in RNase-free water. Libraries were prepared using the mim-tRNAseq workflow (Behrens 2021, 2022) and sequenced on an Illumina NextSeq 500 platform. For V144M/V144M and control samples (experiment 1), libraries were purified by gel size selection prior to sequencing. For A196V/A196L (Patient 10), A196V/A196L (Patient 11) and control samples (experiment 2), the library gel size selection step was omitted.