title: GSM6579141: E7.5_rep2; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted from the epiblast of E3.5, E4.5, E5.5, E6.5, and E7.5 following the manufacturer's protocol (PicoPure RNA isolation kit; KIT0204; ThermoFisher Scientific) and eluted in 7 L elution buffer. The full eluate was utilized to prepare the library. Prior to the construction of the library, the integrity and quality of the RNA were evaluated using a 4200 Agilent TapeStation device and RNA ScreenTape reagents (Agilent). Samples having a RIN > 7.0 for RNA integrity were used. The library was generated with the SMARTer Stranded Total RNA-Seq Kit v3 – Pico Input kit (634485, Takara Bio) following the manufacturer's instructions, with the exceptions listed below. To account for the input differences, fragmentation of RNA was done for 6 minutes at 85 °C on E3.5 and E4.5 epiblast samples, and for 4 minutes at 94 °C on E5.5, E6.5, and E7.5 epiblast samples. Adapters and indices from Illumina were added to distinguish the libraries (SMARTer RNA Unique Dual Index kit, 634451, Takara Bio). Instead of NucleoMag beads, the libraries were purified with AMPure XP beads (A63880, Beckman Coulter). ZapR v3 and R-probes v3 were used to deplete ribosomal cDNA (supplied with the kit). Libraries were amplified for thirteen cycles, purified, and analyzed for quality and concentration using a 4200 Agilent TapeStation instrument and D5000 ScreenTape reagents (Agilent). Accel-NGS Methyl-Seq DNA Library Kit (manufacturer's recommendation)