GSM5388464: GL261 and microenvironment in 2D culture, 3; Mus musculus; RNA-Seq
3D-bioink was cut into 1-2 mm fragments, digested with 600 U/ml collagenase II for 3 h and filtered with a 70 µm mesh prior RNA extraction. Total RNA was then isolated from all samples using EZ-RNA II total RNA isolation kit (Biological industries, California, USA), following the manufacturer's protocol. RNA quality was evaluated by TapeStation® RNA Assay (Agilent Technologies). Libraries were constructed with the NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (#E7760, NEB) using manufacturer's instructions. Twenty-five to 75 ng RNA was amplified using 14 cycles of PCR. Final quality was evaluated by TapeStation® DNA HS Assay (Agilent Technologies). Equimolar pooling of libraries were performed based on Qubit® values and loaded (in triplicates) onto an Illumina® NextSeq 500 platform (Illumina, California, USA) in order to evaluate changes in gene expression between the tested groups.