GSM5015614: Rel1-G01; Homo sapiens; RNA-Seq
Frozen tumor tissue of approximate size 0.2-0.5 cm3 was placed in cold tissue lysis buffer (5M NaCl, 1M MgCl2, 1M Tris buffer (pH 8.0), 10% NP40 (Sigma, USA), 1uM DTT (Invitrogen), 1x Complete protease inhibitor (Roche), 0.4 U/ul RNAseIn (Takara Bio, Japan), 0.2 U/ul SuperaseIn  (ThermoFisher Scientific) and incubated on ice for 15 min with gentle swirling every 2-3 minutes. Then the tissue was triturated, filtered through 40um cell strainer and centrifuged at 1000g for 5 min at 4°C. The sedimented nuclei were resuspended in cold staining buffer (RNAse-free PBS pH 7.4, 1% BSA, RNAseIn, 1M MgCl2, 1mM DTT) and stained with anti-Sox2 antibody (Invitrogen) or respective IgG isotype control (BD), Alexa Fluor 488 Goat anti-Rabbit IgG antibody (Invitrogen) was used as secondary. Nuclei were visualized with 7-AAD (Sigma) staining. Thereafter, Sox2 positive nuclei were sorted a FACS Aria I or III at 4°C in skirted 96-well PCR plates containing 2 ul cell lysis buffer (2U/ul RNAseIn in 0,2% (vol/vol) Triton X-100 (Sigma)). After sorting plates were immediately sealed and placed on dry ice. The library preparation was done using Nextera XT DNA Library Preparation Kit (Illumina) according to the manufacturer instructions, the Illumina Nextera Adapters (sequences provided on http://ngs.biodiv.tw/NGSCore/wp-content/uploads/Documents/illumina-adapter-sequences-1000000002694-08.pdf) were synthetized and HPLC purified in Biomers (http://www.biomers.net/). single cell RNA-seq