title: RNA-seq of in vitro radio-resistant human GBM cell line (LN229) and patient-derived culture (PS1) post YBX3 knockdown
design: RNA-seq of in vitro radio-resistant human GBM cell line (LN229) and patient-derived culture (PS1) post YBX3 knockdown
library_name: PS1 Relapse YBX3sh2 replicate 2_p
library_construction_protocol: Cells were grown to 60% confluence in 10-cm culture dishes, media was removed, and RNA-Isoplus Reagent (Takara Bio) was added to the culture dishes and kept for 2 min on ice for complete cell lysis.  The suspension was collected in a microcentrifuge tube, and RNA was extracted by adding chloroform and precipitating it with isopropanol. RNA samples were quantitated on a Qubit fluorometer, and appropriate dilutions were loaded onto a high-sensitivity RNA screen tape to determine the RNA integrity number (RIN). Samples with 28S/18S peak integrated area ratios of >2 were used for library preparation. 1.5 μg of QC-verified RNA samples was used for poly (A) mRNA isolation from total RNA using the NEB Next Poly (A) mRNA Kit (Cat. no. NEB# 7490), according to the manufacturer's protocol. The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat. no. E7760L) was used to construct double-stranded cDNA libraries from the polyA RNA fraction using RNA fragmentation, first-strand cDNA synthesis with random primers, second-strand cDNA synthesis, end repair of double-stranded cDNA, adapter ligation, removal of excess adapter using sample purification beads, PCR enrichment of adapter-ligated DNA, and clean-up of PCR products as instructed by the manufacturer. The cleaned libraries were quantitated on a Qubit fluorometer, and appropriate dilutions were loaded on a high-sensitivity D1000 screen tape to determine the size range of the fragments and average library size.
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-14224:PS1 Relapse YBX3sh2 replicate 2_p