GSM5226424: 106_V_a Sa2; Homo sapiens; RNA-Seq
For decidualization (DE cells), eSCs from Ctrl (eSCCtrl, n=6) and PCOS (eSCPCOS, n=6) were treated with 8-Br-cAMP (0.5 mM, Sigma-Aldrich, Germany) for 96 h in a low serum medium (3), while pre-decidualised (non-DE cells) were cultured for 96 h using the 0.1% DMSO ( Sigma-Aldrich, Germany) as a vehicle only. Next, DE and non-DE eSCs were treated for 24 h with hormones: non-DE eSCs were post-treated with E2 (10 nM, Sigma-Aldrich, Germany) with or without (±) DHT (100 nM, Sigma-Aldrich, Germany); while the DE eSCs were post-treated with E2 (10 nM, Sigma-Aldrich, Germany) in combination with P4 (E2P4, 1 mM, Sigma-Aldrich, Germany) ± DHT. Total RNA was extracted using RNeasy Mini Kits (Qiagen, Valencia, USA) and RNA sequencing (RNA-seq) libraries were prepared using the SMART-seq2 protocol with modification (Piselli S., 2014). Instead of single cells and original template-switching oligonucleotide (TSO), 12 ng of total RNA for cDNA synthesis and 1 μM TSO (biotin-AAGCAGTGGTATCAACGCAGAGTrGrGrG, Sigma, Schnelldorf, Germany) in combination with 200 nM anchored oligo-T primer (biotin-TTAAGCAGTGGTATCAACGCAGAGTCGACT30+V, where + is LNA nucleotide, Exiqon, Vedbaek, Denmark) was used. Ten cycles of PCR with Phusion High-Fidelity DNA Polymerase (Thermo Scientific, HL, Finland) compatible with the original protocol were used for pre-amplification. 5 µl of cDNA was applied to single-index library preparation using Nextera XT DNA Sample Preparation Kit (Illumina, FC-131-1024, San Diego, CA, USA). AMPure XP beads (Beckman Coulter, San Jose, CA, USA) were used for all clean-up steps. The quality of the resulting libraries was assessed with the 2200 TapeStation system (Agilent, Edinburg, UK). Libraries were pooled and sequenced on NextSeq500 using High Output Flow Cell v2.5 (single-end, 80 bp; Illumina).