GSM6943615: RS243_CD184+_DLL4+; Homo sapiens; RNA-Seq
On day 8 of culture, embryoid bodies were dissociated with 0.25% Trypsin-EDTA for 8 minutes and Collagenase II for 30 minutes, immunolabled at 4C for 25 minutes. CD34+CD32+CD43–CD184–CD73– and CD34+CD184+CD73+DLL4+CD43neg  cells were then FACS sorted using a BD FACS Aria Fusion II and stored on ice until ready for RNA extraction. We extracted total RNA using the ReliaPrep RNA Cell Miniprep System (Promega). We quantified RNA concentration using Qubit RNA HS Assay Kit using Qubit 3.0 and we evaluated RNA integrity with Agilent RNA 6000 Pico Kit using Bionalyzer (Agilent). RNA-Seq libraries were generated using the Smart-seq2 method (Picelli et al., 2014) starting from 0,876 ng of RNA. Retro-transcription was performed using SuperScript II Reverse Transcriptase, cDNA was PCR-amplified (16 cycles) with KAPA HiFi HotStart and purified with AMPure XP beads. After purification, we determined cDNA concentration using Qubit dsDNA HS Assay Kit at Qubit 3.0 and we assessed size distribution at Agilent 4200 TapeStation system. We performed the tagmentation reaction starting from 0.5 ng of cDNA for 30 minutes at 55°C and we performed enrichment PCR using 12 cycles. Libraries were purified with AMPure XP beads, quantified using Qubit 3.0, assessed for fragment size distribution on an Agilent 4200 TapeStation system.