GSM9225783: RIF endometrium-on-a-chip 3; Homo sapiens; RNA-Seq
Total RNA from samples was extracted using Labozol reagent per sample. After adding chloroform and thorough mixing, the tubes were centrifuged at 13,000 rpm for 20 min at 4 °C. The aqueous phase was transferred to new tubes, and an equal volume of isopropanol was added, followed by centrifugation at 13,000 rpm for 30 min at 4 °C. Pellets were washed with 70% ethanol. RNA pellets were dissolved in 20 μL of nuclease-free water. RNA quality was evaluated using a NanoDrop spectrophotometer, and only samples with 260/280 and 260/230 absorbance ratios between 1.9 and 2.2 were used for RNA-seq analysis. For control and test RNAs, the construction of library was performed using QuantSeq 3' mRNA-Seq V2 Library Prep Kit FWD (Lexogen) according to the manufacturer's instructions. In brief, each total RNA were prepared and an oligo-dT primer containing an Illumina compatible sequence at its 5' end was hybridized to the RNA and reverse transcription was performed. After degradation of the RNA template, second strand synthesis was initiated by a random primer containing an Illumina compatible linker sequence at its 5' end. The double stranded library was purified by using magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The finished library is purified from PCR components. High-throughput sequencing was performed as single-end 100bp sequencing using NextSeq 2000 (Illumina Inc.).