title: GSM5969813: Input DNA in EB+48hr(iAscl1.Lmx1b.Foxa2.V5.TRE.Pet1.HA); Mus musculus; ChIP-Seq
library_construction_protocol: Cells were collected and fixed with 1 mM DSG (ProtoChem) followed by 1% FA (vol/vol) each for 15 min at room temperature. Pellets containing 25–30 × 10^6 cells were aliquoted and flash-frozen at −80 °C. Cells were lysed in lysis buffer containing 50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol) with 1× protease inhibitors (Roche, 11697498001) at 4 °C for 10 minutes. Cells were resuspended in 50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol), and incubated 10 minutes at 4 °C. Nuclear extracts were resuspended in cold sonication buffer (50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate (wt/vol), 0.1% SDS (wt/vol)). Sonication was performed with Bioruptor Pico sonicator device (Diagenode) with 30 sec ON/ 30 sec OFF, 18 cycles, with Bioruptor sonication beads (0.45 mg beads per 1 ml sample). Immunoprecipitation was done overnight at 4 °C on a rotator with Dynabeads protein-G (Thermo Fisher) conjugated antibodies. 5 µg of the following antibodies were used for immunoprecipitation: anti-Ascl1 (Abcam, ab74065); anti-HA (Abcam, ab9110); anti-V5 (Abcam, ab15828). Subsequent washes were done in 1X sonication buffer (cold) first, sonication buffer with 500 nM NaCl (cold), LiCl wash buffer (20 mM Tris-HCl (pH 8.0)) (cold), 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) (cold), and TE buffer (10 mM Tris, 1 mM EDTA, (pH 8)) (cold). Samples were eluted in elution buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 1% SDS) by incubating for 45 min at 65 °C. Eluted sample and input (sonicated only) were incubated overnight at 65 °C to reverse the crosslink. RNA was digested by the addition of 0.2 mg ml–1 RNase A (Sigma) and incubating for 2 h at 37 °C. Protein digestion was performed by adding 0.2 mg ml–1 Proteinase K (Invitrogen) for 30 min at 55 °C. DNA extraction was done with Phenol:chloroform:isoamyl alcohol (25:24:1; vol/vol) (Invitrogen) followed by ethanol precipitation. 1/3 of ChIP DNA (1:100 dilution of input DNA) was used to prepare lllumina DNA sequencing libraries. Bioo Scientific multiplexed adapters were ligated after end repair and A-tailing, and unligated adapters were removed with Agencourt AmpureXP beads (Beckman Coulter) purification. Adapter-ligated DNA was amplified by PCR using TruSeq primers (Sigma). DNA libraries between 300 and 500 bp in size were purified from agarose gel using a Qiagen minElute column, and the final quantification of the library before pooling was done using a KAPA library amplification kit (Roche Lightcycler 480). The libraries were sequenced on an Illumina NextSeq 500 using V2 chemistry for 50 cycles (single-end) at NYU Genomics Core facility.