title: GSM8454207: fem_line,rep1,d30; Homo sapiens; RNA-Seq
library_construction_protocol: The RNA was extracted from the iCMs on day four and day 30. Cells were first washed with PBS and dissociated from the plate either by 3 minutes incubation in 37°C with PBS with EDTA (4 days iCMs) or 5-10 minutes incubation with TrypLE™ (Thermo Fisher Scientific 12604013) (30 days iCMs). Dissociated cells were washed with medium from the plate and collected in 15ml falcon tubes. The centrifugation for 3min/1300rpm/RT allowed us to obtain the cell pellet. The supernatant was discarded, and the cell pellet was washed with cold PBS twice and centrifuged at 4min/2000rpm/4°C. Finally, the cell pellet was resuspended in 1ml of TRIzol™ Reagent (Thermo Fisher Scientific 15596026) or 200µl of RNAlater (Sigma-Aldrich R-0901) and stored at -20°C for further RNA extraction. Standard phenol/chloroform protocol was used to isolate RNA from TRIzol™ Reagent samples. Total RNA isolation from RNAlater collected samples was conducted with mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific AM1561). The obtained RNAs' amount and quality were measured on a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Scientific). The 500ng of RNA was used as a template to generate complementary DNA with SuperScript™ IV First-Strand Synthesis System with ezDNase™ Enzyme (Thermo Fisher Scientific 18091150). Reverse transcription was conducted using a thermal cycling program: 23°C/10 minutes, 55°C/10min, and 80°C/10 min on T100 Thermal Cycler (Bio-Rad). For each sample, RNA libraries were prepared from 100 ng RNA using the KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems) according to manufacturer's protocol, followed by quality check using Fragment Analyzer (Agilent). The libraries were normalized and pooled, and then sequenced using NovaSeq6000 platform (Illumina) to an average of 120 M 151 bp paired-end reads per sample.