Illumina HiSeq 4000 sequencing; ChIP-Seq of ES-E14 mouse embryonic stem cells investigating changes in Sall4
ChIP-Seq of ES-E14 mouse embryonic stem cells investigating changes in Sall4
For each ChIP, 25x106 ES-E14 ESCs were plated into 15cm dishes the day before the experiment. Cells were crosslinked at 37°C for 10min with 1% formaldehyde. Following quenching for 5min at room temperature with 125mM glycine, cells were washed 3 times with ice-cold PBS. 10ml of swelling buffer (10mM KCl, 1.5mM MgCl2, 25mM HEPES pH7.9, 0.1% NP-40) freshly supplemented with 1x protease inhibitor cocktail (Roche ref. 11873580001) was added into each plate, followed by a 10min incubation at 4°C. Nuclei were collected by scraping and transferred into 15ml tubes. Samples were centrifuged at 4°C for 5min at 3,000rpm and the supernatant was removed. Crosslinked nuclei were quickly frozen on dry ice and stored at -80°C before proceeding with the rest of the protocol. Crosslinked nuclei were thawed on ice, resuspended in 2ml of sonication buffer (140mM NaCl, 1mM EDTA, 1% Triton, 0.1% Na-deoxycholate, 0.1% SDS, 50mM HEPES pH7.9) freshly supplemented with 1x protease inhibitor cocktail, and transferred into 1.5ml TPX tubes (Diagenode). Chromatin was sonicated by performing 20x sonication cycles (30sec on/ 30sec off) using the Bioruptor Twin instrument (Diagenode) with a 4°C water bath. Samples were centrifuged at 4°C for 30min at 13,000rpm to remove insoluble material. Supernatants (soluble chromatin fraction) were collected and transferred into 1.5ml LoBind tubes (Eppendorf). To evaluate the amount of chromatin in each sample, a 2µl aliquot was alkaline-lysed with 0.1M NaOH and measured using a micro-volume spectrophotometer (Nanodrop ND-1000). For each immunoprecipitation, 700µg of chromatin was mixed with 5µg of anti-SALL4 antibody (Santa Cruz ref. sc-101147 or Abcam ref. ab29112) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail. Sall4 knockout ESCs chromatin samples were used as a negative control. Samples were incubated overnight at 4°C on a rotating wheel. 50µl of either Protein A (ChIP with Abcam ref. ab29112) or Protein G (ChIP with Santa Cruz ref. sc-101147) magnetic beads (Invitrogen Dynabeads), previously equilibrated in sonication buffer, was added into each sample. Following a 3h incubation at 4°C on a rotating wheel, beads were extensively washed with 1ml of each of the following buffer: 1x with sonication buffer, 1x with wash buffer A (500mM NaCl, 1mM EDTA, 1% Triton, 0.1% Na-deoxycholate, 0.1% SDS, 50mM HEPES pH7.9), 1x with wash buffer B (250mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% Na-deoxycholate, 20mM Tris pH8.0), 2x with TE buffer (Sigma-Aldrich ref. 93283). Between each wash, beads were incubated for 5min at room temperature on a rotating wheel. Finally, DNA was eluted by resuspending beads in 250µl of elution buffer (50mM Tris pH7.5, 1mM EDTA) freshly supplemented with 1% SDS, and by incubating samples at 65°C for 5 min. Samples were further incubated for 15min at room temperature on a rotating wheel and the supernatant (eluted chromatin) was collected into a new 1.5ml LoBind tube. The elution was repeated a second time to obtain 500µl of immunoprecipitated chromatin. To extract DNA from immunoprecipitated chromatin or from the input material (50µl of soluble chromatin), crosslinking was reversed by incubating samples overnight at 65°C in a total volume of 500µl with 160mM NaCl and 20μg/ml RNase A. Then, 5mM EDTA and 200μg/ml Proteinase K were added to the samples, followed by a 2h incubation at 45°C. Finally, DNA was purified by phenol-chloroform extraction (Invitrogen ref. 15593031) followed by ethanol precipitation with 2x volumes of 100% ethanol, 0.1x volume of 3M sodium acetate, and 40µg of glycogen (Invitrogen ref. 10814010). Samples were incubated at -80°C for at least 1h and centrifuged at 4°C for 30min at 13,000rpm. The supernatant was removed and DNA pellets were washed with 70% EtOH. Following a final spin at 4°C for 15min at 13,000rpm, DNA pellets were air dried and resuspended in 30-100µl TE buffer (Sigma-Aldrich ref. 93283) or H2O. DNA concentration was quantified using the Qubit dsDNA HS Assay Kit (Invitrogen ref. Q32854). ChIP-seq libraries were prepared using the KAPA Hyper Prep Kit (Roche ref. 07962347001) together with KAPA dual-indexed adapters (Roche ref. 08278555702), following manufacturer's instruction. ChIP-seq libraries were quantified using the Qubit dsDNA HS Assay Kit (Invitrogen ref. Q32854) and fragment size was evaluated using the Agilent 2100 Bioanalyzer (Agilent High Sensitivity DNA Kit).