title: GSM9534872: Suspicion of Ewing's sarcoma; Homo sapiens; OTHER
library_construction_protocol: DNA were extracted from frozen tumor samples using the standard phenol-chloroform procedure. Remaining of the DNA was extracted using the Maxwell automat. The quality and quantity of DNA were assessed using Nanodrop (Thermo Fisher Scientific) and Qubit (Thermo Fisher Scientific). The extracted DNA was quantified using the Qubit Fluorometer (Quant-iT dsDNA BR Assay Kit; Thermo Fisher Scientific) and qualified using TapeStation (Genomic DNA screenTape; Agilent) following the manufacturer's instructions. DNA libraries were prepared from 2 µg of genomic DNA, quantified using the Qubit assay. Two libraries, P6 and P7, were generated from 343 ng and 987 ng of DNA, respectively, based on available quantities. The library preparation process is efficient, requiring approximately half a day to complete. The main steps include mechanical DNA fragmentation using Covaris® g-tubes to achieve a target fragment size of 20 kb, followed by end repair, adapter ligation, and library purification. Initially, libraries were prepared using the ligation sequencing kit LSK-SQK110 and later with LSK-SQK114 (Oxford Nanopore Technologies) due to a chemistry update during the study, following the manufacturer's guidelines. Sequencing was performed in two batches using R9 or R10 flow cells. First, half of the library was loaded onto a MinION MK1B device for approximately 24 hours of sequencing. Once data acquisition reached a plateau and most flow cell pores became saturated, sequencing was paused. The flow cell was then washed with a nuclease mix to recover as many active pores as possible before loading the remaining half of the library for an additional 24 hours of sequencing.