Sc11198x

Cell line chromatin was prepared as previously described (33 (link), 48 (link), 49 (link)). Embryonic chromatin was prepared using E18.5 wildtype CD1 embryos. 8–12 whole pancreata per prep were microdissected, flash frozen, sheared (22g needle), and solution crosslinked using 2% formaldehyde in PBS for 10 min at room temperature. Chromatin fragments were generated by sonication using the Diagenode Bioruper Pico and precleared using Protein-G Dynabeads (10004D; Invitrogen). ChIP was then performed using EZ-Magna ChIP A/G Kit (Millipore, #17–10086). Briefly, chromatin aliquots were incubated with A/G beads and anti-Ldb1 (ab96799; Abcam or sc11198x; Santa Cruz) or rabbit IgG (P120–101; Bethyl Laboratories) antibodies at 4°C overnight with rotation. Bound complexes were washed and crosslinks were reversed, DNA was purified. qPCR was performed on immunoprecipitated DNA using SYBR Green PCR master mix (Bio-Rad) and primers for Pdx1 (19 (link)) and Ngn3 (24 (link)) regulatory sequences (Supplemental Table 2). For cell line ChIP, enrichment of target control sequences in ChIP DNAs was normalized to inactive albumin control sequences (50 (link)) and calculated relative to rabbit IgG enrichment set as one-fold (ΔΔCt). Enrichment of target regions for in vivo ChIP was calculated as percent input (as described (51 (link))) and plotted relative to rabbit IgG enrichment (set to one-fold).