GSM7695856
RNA was isolated from the biopsies preserved in RNAlater by using a spin column-based nucleic acid purification approach. Frozen specimens were dissected free of chorionic and basal plates, which were inconsistently present in the samples. Then they were placed in 2 mL tubes with 2.8 mm ceramic beads (Fisherbrand) and 600 µl lysis buffer (RLTplus; Qiagen), containing 1% (v/v) beta-mercaptoethanol (Sigma-Aldrich) and 0.5% (v/v) Reagent DX (Qiagen). Then, samples were homogenized at 4˚C using a Bead Mill 24 device (Fisherbrand), which enabled delivery of two 20 sec pulses with a 10 sec rest in between. Debris was pelleted by centrifugation at 15,000 xg for 2 min (Eppendorf 5415 R microcentrifuge). The supernatants were transferred to 1.5 mL tubes (Eppendorf) and the centrifugation step was repeated. Total RNA was purified following the manufacturer's protocol (Qiagen RNeasy Plus Mini Kit). Absorbance at 280, 260 and 230 nm was measured (Nanodrop 2000; Thermo Scientific) to determine concentration and purity; 5 µL was aliquoted to measure integrity, and the remaining sample was stored at -80˚C.  The integrity of the RNA  was determined via microfluidic electrophoresis (Agilent Tapestation 4200) using either an RNA ScreenTape Assay or High Sensitivity ScreenTape Assay, depending on the concentration. Aliquots with concentrations exceeding the dynamic range were diluted to 500 ng/uL immediately prior to the assay, which was performed using the manufacturer's protocol. A RNA Integrity Number (RIN) was automatically assigned on a 0-10 scale according to a standardized assessment of the electrophoretic trace of the 18S and 28S rRNAs. Samples having a RIN ≥ 7 were considered suitable for sequencing (RNA-Seq). A portion of each sample (500 ng) was used for cDNA synthesis and library construction. A genomic DNA cleanup step was performed to remove contaminants. Next, libraries were prepared using the Kapa Biosystems adapter kit. RNA sequencing was performed on a 150PE NovaSeq S4 flowcell to obtain ~25 million reads per sample.