GSM3145236: GFP_low_2w2w_Sample2; Homo sapiens; RNA-Seq
GBM cells that were grown as 3D organoids were gently triturated with Pasteur pipettes of decreasing tip diameter. Cells were pelleted and resuspended in FACS buffer, and debris was removed by pelleting cells at 155 g for 6 min through a phase of 2% BSA in Hibernate-E LF. Cells were resuspended in FACS buffer and passed through a 70 µm mesh filter. DAPI (Invitrogen) was added to cell suspensions at a concentration of 5 µg/ml to stain dead cells. Cell suspensions were analyzed by flow cytometry (BD LSRII) or sorted by FACS (BD FACSAria IIu), and data was evaluated with FACSDiva and FlowJo software. RNA from FACS sorted GBM cells (GFP-high and -low) was isolated with RNeasy (QIAGEN), and cDNA libraries for Illumina next-generation sequencing were either prepared with NEBNext Ultra Directional RNA Library Prep Kit (NEB E7420) for 2wk2wk samples, or with NuGEN Ovation amplification followed by NEBNext Ultra DNA Library Prep Kit (NEB 7370) for 2wk4wk samples. Sequencing was performed on Illumina HiSeq2500 devices in rapid run mode with paired 50 bp reads.
GEO Accession
GSM3145236