title: GSM6638194: MV3, day2, core, replicate2; Homo sapiens; RNA-Seq
library_construction_protocol: MV3 H2B-GFP spheroids with a diameter of 1 mm (50,000 cells) were cultured for two days. Higher number of cells was required to obtain enough material for RNA sequencing. After replacing the medium by PBS, samples were transferred to a sterile fluorescence stereomicroscope for image guided dissection (Leica MZFLII, 0.125 NA., EL6000external light source, GFP Plus filter sets). PBS was removed and the core was punched out with a 1 mm biopsy puncher (Miltex, Sterile Biopsy punch with plunger, #3331AAP/25) by pressing the punch down around the spheroid core and sucking it out using the punch plunger. The core was released into a sterile 1.5 ml Eppendorf tube and placed on dry ice for snap-freezing. The corresponding invasion zone was controlled on any left-over core region, recognizable by much brighter fluorescence and removed from the collagen gel with a 3 mm biopsy puncher (Miltex, Sterile Biopsy punch with plunger, #3332P/25). Exclusion criteria were ellipsoid shaped spheroids and unsuccessfully punched spheroids with ruptured gels. Samples were stored at -80°C until RNA extraction. RNA was isolated using an RNA isolation kit (Qiagen, RNeasy Micro Kit) according to the manufacturer's protocol. RNA concentration was measured using a NanoDrop 2000 (ThermoFisher) and stored at -80°C. The integrity and quality of RNA was evaluated using the Experion RNA HighSens AnalysisKit (Biorad, 7007105) on a 2100 Bioanalyzer platform (Agilent). For CDNA synthesis starting from 1-10 ng total RNA, SMARTer Ultra low Input RNA kit (Clontech, 634936) was used and processed according to the manufacturer's protocol. The transcribed DNA was sheared into fragments of 200-600 bp using Bioruptor Pico sonication device (Diagenode). Subsequently the library preparation for Next Generation Sequencing has been carried out according to the Illumina standard protocol using the KAPA Hyper prep kit (KAPA Biosystems).