title: NextSeq 500 sequencing; ChIP-seq of PBX1, PBX2 and LEF1 in wild-type and Pbx1/Pbx2 double-knockout EpiSCs differentiated in vitro to pre-somitic mesoderm in the first 48 hours of differentiation.
design: ChIP-seq of PBX1, PBX2 and LEF1 in wild-type and Pbx1/Pbx2 double-knockout EpiSCs differentiated in vitro to pre-somitic mesoderm in the first 48 hours of differentiation.
library_construction_protocol: EpiSCs were seeded at ~2 x 10^3 cells/cm2 in EpiSC medium in 150 mm tissue-culture dishes (Corning, 430599) pre-coated with 15 µg/ml human Fibronectin (Merck Millipore, FC01015), and differentiated to pre-somitic mesoderm (PSM) as described below. At desired time-points, cells were washed with PBS and fixed directly on the plate by the addition of 20 ml of PBS containing 1% (v/v) Formaldehyde Fixative Ultrapure EM Grade (Rockland, KHF001) for 8-10 min at RT. Fixation was stopped by 5 min incubation with 125 mM glycine at RT. After 2 washes with cold PBS, fixed cells were harvested in lysis buffer (50 mM Tris-HCl pH 8.1, 100 mM NaCl, 5 mM EDTA, 0.2% (w/v) NaN3, 0.5% (w/v) SDS, supplemented with 1x Protease inhibitor cocktail (Roche, 11836170001) and 1 mM PMSF) by extensive scraping using a pipette tip. Following centrifugation at 1,000 x g for 5 min at 4°C, the pellet was resuspended in 1 ml of FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl, supplemented with 1x Protease inhibitor cocktail) per 0.5 ml of pellet. 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). 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. Chromatin was disrupted by sonication using a chilled Bioruptor Plus sonicator UCD-300 (Diagenode) for 8-12 pulses (cycles of 30 s on / 45 s off, high setting at 4°C) to obtain fragments of 200-500 bp in size. The extract was spun for 30 min at 20,000 x g at 4°C, and the soluble fraction was aliquoted and stored at -80°C until use. ChIP was performed with a protocol modified from that of Kolasinska-Zwierz P. et al., Nat Genet 41, 376-381 (2009). For each IP, 20 μg of chromatin were incubated overnight in 1 ml of FA buffer containing 1% N-lauroylsarkosine sodium salt with the following antibodies: PBX1 (polyclonal, Rabbit; Cell Signaling Technology, cat. nr. 4342, 1:100); PBX2 (polyclonal, Rabbit; Thermo Fisher Scientific, cat. nr. PA5-63964; 1:125); LEF1 (monoclonal, Rabbit; Cell Signaling Technology, cat. nr. 2230; 1:100). 10% of the IP volume of the chromatin was retained as an input reference. After overnight rotation at 4°C, 30 μl of washed and blocked magnetic Protein A (Thermo Fisher Scientific, 10006D) or Protein G Dynabeads (Thermo Fisher Scientific, 10003D) were added and the incubation continued for 2 additional h. Beads were extensively washed at RT as described in Kolasinska-Zwierz P. et al. DNA was eluted twice with 57 μl of elution buffer (1% (w/v) SDS in TE pH 8.0 with 250 mM NaCl) at 65°C, 15 min each time. Eluted DNA was incubated with 20 μg of RNase A for 30 min at 37°C and then with 20 μg of Proteinase K for 1 h at 55°C. Input DNA was also diluted in 114 ul of elution buffer and treated as ChIP samples. Crosslinks were reversed at 65°C overnight and DNA was purified using the QIAGEN MinElute PCR Purification Kit (QIAGEN, 28006) according to the manufacturer's instructions. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (New England BioLabs, E7645S) as per manufacturer's instructions. Libraries were amplified for 5–7 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).