title: NextSeq 500 sequencing; RNA-seq of wild-type, Pbx1/Pbx2 double-knockout and pMsgn1-mut EpiSCs differentiated in vitro to pre-somitic mesoderm in the first 24 hours of differentiation.
design: RNA-seq of wild-type, Pbx1/Pbx2 double-knockout and pMsgn1-mut EpiSCs differentiated in vitro to pre-somitic mesoderm in the first 24 hours of differentiation.
library_construction_protocol: EpiSCs were seeded at ~6 x 10^3 cells/cm2 in 6-well cell-culture plates (Corning, 3506) pre-coated with 15 µg/ml human Fibronectin (Merck Millipore, FC01015), and differentiated to pre-somitic mesoderm (PSM) as described below. Wild-type EpiSC lines were derived from E6.5 mouse embryos (129 background) and maintained in 6-well cell-culture plates (Corning, 3506) pre-coated with 15 µg/ml human Fibronectin (Merck Millipore, FC01015) in N2B27 medium (prepared as described in Ying Q.L. & Smith A.G., Methods Enzymol 365, 327-341 (2003)), supplemented with 20 ng/ml Activin A, 10 ng/ml bFGF and 1 µM XAV939 (EpiSC medium). Pbx1/Pbx2 double-knockout (Pbx1/2-DKO) EpiSC lines and lines carrying base-pair substitutions on the Msgn1 promoter that abolish PBX recruitment (pMsgn1-mut) were generated by CRISPR/Cas9. All differentiation was conducted in serum-free, feeder-free and monolayer conditions in chemically-defined N2B27-based media. Culture conditions followed a modified protocol from Loh K.M. et al., Cell 166, 451-467 (2016). EpiSCs were seeded at ~6 x 10^3 cells/cm2 in 6-well cell-culture plates pre-coated with 15 µg/ml human Fibronectin, and cultured in EpiSC medium for 24 h. EpiSCs were first differentiated towards anterior primitive streak (APS) using 30 ng/ml Activin A, 4 µM CHIR-99021, 20 ng/ml bFGF and 100 nM PIK-90 (APS medium) for 12 h, and then to pre-somitic mesoderm (PSM) using 1 µM A-83-01, 3 µM CHIR-99021, 250 nM LDN193189 and 20 ng/ml bFGF (PSM medium) for 36 h. At desired time-points (12 hours and 24 hours of PSM differentiation), cells were lysed and total RNA was extracted using the RNeasy Mini Kit (Qiagen, 74106) according to the manufacturer's instructions. RNA concentration was quantified by NanoDrop (Thermo Fisher Scientific) and quality was verified with a Fragment analyzer (Agilent). Library preparation was carried out using 0.5 μg of RNA with NEBNext Ultra II RNA library Prep Kit (New England BioLabs, E7770S) as per manufacturer's instructions. Libraries were amplified for 4 total PCR cycles and purified with AMPure XP beads (Beckman Coulter Life Sciences, A63881). DNA concentration was measured with a Qubit Fluorometer (Thermo Fisher Scientific, Q32854) and fragment size was determined with a Fragment analyzer (Agilent).