NextSeq 500 paired end sequencing; ATAC-seq of wild-type, Pbx1/Pbx2 double-knockout and pMsgn1-mut EpiSCs differentiated in vitro to pre-somitic mesoderm in the first 48 hours of differentiation.
ATAC-seq of wild-type, Pbx1/Pbx2 double-knockout and pMsgn1-mut EpiSCs differentiated in vitro to pre-somitic mesoderm in the first 48 hours of differentiation.
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 (EpiSCs, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours of differentiation), 50,000 cells per replicate were transposed as described in Buenrostro J.D.et al., Nat Methods 10, 1213-1218 (2013), with modifications based on Corces M.R. et al., Nat Methods 14, 959-962 (2017). Libraries were generated using the Ad1_noMX and Ad2.1–2.16 barcoded primers described in Buenrostro J.D.et al., Nat Methods 10, 1213-1218 (2013), and amplified for 10 total PCR cycles. Libraries were purified with AMPure XP beads (Beckman Coulter Life Sciences, A63881) to remove contaminating primer dimers and fragments larger than 1,000 bp. Library quality was assessed using the Fragment analyzer (Agilent) and quantitated by Qubit assay (Thermo Fisher Scientific, Q32854).