RNA-seq for identifying semi-extractable and OOPS RNAs in naive and primed mouse embryonic stem cells
RNA-seq for identifying semi-extractable and OOPS RNAs in naive and primed mouse embryonic stem cells
For control and semi-extractability samples: naive pluripotent stem cells (nPSCs), primed (pPSCs) and 1-day Wnt-differentiated cells (dPSC) were washed twice with PBS, then lysed by adding TRIzol to the plate and scraping the cells off. The samples were snap frozen on dry ice and stored at -80°C prior isolation. For OOPS, one confluent 10 cm plate was used per replicate, washed twice with ice-cold PBS before crosslinking them at 400 mJ/cm2 (UV 254 nm). After removing residual PBS, 1 mL TRIzol-LS was added to the plate before scraping off the cells content and transferring it to a 1.5 mL Eppendorf tube that was stored at -80°C.  Naive mESC (nPSCs) were grown in 2i conditions (PMID: 18497825), primed (pPSCs) in N2B27 basal medium supplemented with ActA 20 ng/ml, bFGF 12 ng/mL, IWP2 2 µM, and dPSCs were acquired from pPSCs, by switching the medium to N2B27 basal medium supplemented with ActA 20 ng/mL, bFGF 12 ng/mL, Wnt3a 250 ng/mL for 24 hours before collecting them.  For control: TRIzol samples were thawed and RNA was extracted from the aqueous phase, following manufacturer's instructions.  For semi: TRIzol samples were thawed and heated for 10 min at 55°C and also passed 40 times through a 20G needle (PMID: 28404604).  For OOPS: RNA was isolated from the interphase following published protocol (PMID: 30607034, PMID: 32651564).  Libraries for sequencing were prepared using CORALL Total RNA-Seq with RiboCop rRNA depletion verion 1 (Lexogen) with 500 ng DNase-treated RNA as input.