Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: [Samples E14.5 and E12.5] Single cell suspensions of embryonic cells were fixed for 10 min with 1% PFA at RT, washed twice with PBS and stained with PerCP-eFluor® 710–conjugated anti-EpCAM 1:400 (eBioscience), PE-conjugated anti-CD45 at 1:400 (BD Biosciences) and anti-CD31 at 1:400 (BD Biosciences) antibody for 15-30 min at RT. Two hundred fifty thousand EpCAM+ embryonic intestinal cells, Lgr5-EGFPhigh ISCs or enterocytes were isolated by FACS and stored at -80°C. To obtain fragments around 350-700 bp, chromatin was resuspended in 150 uL of sonication buffer (1% SDS, 50 mM Tris-Cl pH 8.0, 10 mM EDTA) and sonicated for 56 cycles 10 sec ON/ 90 sec OFF (Diagenode) at 4C.H3K27me3 (Millipore #17-622) antibodies were used. 20 uL of Protein A/G sepharose beads (Sigma, P6486 and E3403) were pre-incubated for 3 hours with 0.5 ug of antibody in RIPA buffer, then washed3 times with RIPA buffer. The sonicated chromatin was diluted till final volume 750 uL and incubated with antibody-bead slurry at 4C overnight. Beads were washed 3 times with 1 mL of RIPA buffer and once with TE. DNA was eludet and incubated with RNAse A for 30 min, followed by ON incubation with PK. DNA was extracted using phenol: chloroform. [Samples AE and ISC] Single cell suspensions of adult cells were fixed for 10 min with 1% PFA at RT, washed twice with PBS and stained with PerCP-eFluor® 710–conjugated anti-EpCAM 1:400 (eBioscience), PE-conjugated anti-CD45 at 1:400 (BD Biosciences) and anti-CD31 at 1:400 (BD Biosciences) antibody for 15-30 min at RT. Two hundred fifty thousand EpCAM+ embryonic intestinal cells, Lgr5-EGFPhigh ISCs or enterocytes were isolated by FACS and stored at -80°C. To obtain fragments around 350-700 bp, chromatin was resuspended in 150 uL of sonication buffer (1% SDS, 50 mM Tris-Cl pH 8.0, 10 mM EDTA) and sonicated for 56 cycles 10 sec ON/ 90 sec OFF (Diagenode) at 4C. H3K27me3 (Millipore #17-622) antibodies were used. 20 uL of Protein A/G sepharose beads (Sigma, P6486 and E3403) were pre-incubated for 3 hours with 0.5 ug of antibody in RIPA buffer, then washed3 times with RIPA buffer. The sonicated chromatin was diluted till final volume 750 uL and incubated with antibody-bead slurry at 4C overnight. Beads were washed 3 times with 1 mL of RIPA buffer and once with TE. DNA was eludet and incubated with RNAse A for 30 min, followed by ON incubation with PK. DNA was extracted using phenol: chloroform. Libraries were prepared using NuGEN Ovation Ultralow Library System V2 (NuGEN) according to manufacturer’s instructions.