title: GSM2124249: ChIP-seq Onecut2 in EB+48hrDox(iNgn2.Isl1.Lhx3.V5) replicate 1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were collected at different time points after NIL induction and fixed with 1 mM DSG (ProteoChem) followed by 1% formaldehyde (vol/vol) for 15 min at 20–25°C. Pellets containing ~25 × 10^6 cells were stored at −80 °C. Cells were thawed on ice, resuspended in 5 ml of Lysis Buffer  A (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 for 10 min at 4°C in a rotating platform. Samples were spun down for 5 min at 1,350 g, resuspended in 5 ml Lysis Buffer B (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) and incubated for 10 min at 4 °C in a rotating platform. Samples were spun down for 5 min at 1,350 g, resuspended in 3 ml of Sonication Buffer (50 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Trition X-100, 0.1% sodium deoxycholate (wt/vol), 0.1% SDS (wt/vol)). Nuclear extracts were sonicated on ice using a Branson 450 Sonifier (20% power amplitude; 18 cycles x 30 seconds pulses with 1 minute interval between each pulse) to sheer cross-linked DNA to an average fragment size of approximately 500 bp. Sonicated chromatin was incubated for 16 h at 4 °C with Dynabeads protein-G (Life Techologies) conjugated with either rabbit polyclonal antibody to V5 (ab15828, Abcam) to immunoprecipitate Lhx3 or a  combination of monoclonal antibodies (40.3A4, 39.3F7 and 40.206, DSHB) for Isl1. Ngn2 was immunoprecipitated by using a goat polyclonal antobody (sc-19233, Santa Cruz), whereas to immunoprecipitate Ebf2 and Onecut2, a rabbit polyclonal antibody (ab156999, Abcam) and a sheep polyclonal antibody (AF294, R&D Systems) were used, respectively. For histone modifications the following rabbit polyclonal antibodies were used: ab8895 (Abcam) for H3K4me1, ab7766 (Abcam) for H3K4me2, 39159 (Active Motif) for H3K4me3, ab4729 (Abcam) for H3K27ac and 39155 (Active Motif) for H3K27me3. After incubation, and with the aid of a magnetic device, beads were washed once with Sonication Buffer, than with Sonication buffer and 500 nM NaCl and once with LiCl Wash Buffer (20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and 1 ml of TE (10 mM Tris, 1 mM EDTA, pH 8). Then, beads were centrifuged at 950 g for 3 min and the residual TE removed with a pipette. 210 µl of Elution Buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 1% SDS) was added to the beads followed by incubation at 65°C for 45 min with a brief pulse of vortex every 10 min. 200 µl of supernatant was removed after a 1 min centrifugation at 16,000 g. The crosslink was reversed by 16 h incubation at 65°C. RNA was digested by the addition of 200 µl of TE and RNAse A (Sigma) at a final concentration of 0.2 mg/ml and incubated for 2 h at 37°C. Protein was digested by the addition of Proteinase K (Invitrogen) at a final concentration of 0.2 mg/ml, supplemented with CaCl2 followed by a 30-min incubation at 55°C. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1; vol/vol) (Invitrogen) and then recovered with an ethanol precipitation with glycogens as carrier. The pellets were suspended in 70 µl of water. One third (24ul) of ChIP DNA was used to prepare llumina DNA sequencing libraries. Briefly, after end repair and A-tailing, Illumina-compatible Bioo Scientific multiplexed adapters were ligated and the unligated adapters removed through purification using Agencourt AmpureXP beads (Beckman Coulter). Adapter-ligated DNA was amplified by PCR using TruSeq Fw primer (AATGATACGGCGACCACCGAGATCTACAC) and TruSeq Rev primer (CAAGCAGAAGACGGCATACGAGAT), both from Sigma. DNA libraries between 300 and 500 bp in size were gel purified. KAPA SYBR FAST Roche LightCycler 480 2X qPCR Master Mix (Kapa Biosystems) was used in 20 μl reactions that were analyzed in a Roche LightCycler. Fifty base pair single-end sequencing was performed using Illumina HiSeq-2500 at the NYU Genome Core facility.
attributes.tag: GEO Accession
attributes.value: GSM2124249