GSM5484530: JT_500_0h_ixen_Nanog_ChIP_merged_rep2; Mus musculus; ChIP-Seq
30 million cells were fixed by resuspending them in 1% formaldehyde in HBSS (1ml/million cells). Cells were incubated for 10 mins at room temperature, protected from light, on a nutator. After 10 mins, 1.42M glycine (100µl/ml of fixative) was added and cells were mixed by inversion and incubated for 5 mins at room temperature to quench PFA. Cells were then pelleted at 2000g, at 40C for 5 mins and washed twice in 1X TBS (50 mM Tris-Cl, pH 7.5, 150 mM NaCl). Cell pellets were then flash frozen and stored at -800C. To perform ChIP, pellets were thawed on ice for 10 mins and lysed in Farnham lysis buffer (5mM PIPES PH 8.0, 85mM KCl, 0.5% NP-40, supplemented with protease inhibitors). To lyse cells, pellets were reconstituted in 1ml buffer/ 10 million cells and incubated on ice for 10 mins, following which they were dounced and then centrifuged at 2000g, at 40C for 5 mins. The pelleted nuclei were then resuspended in 500µl RIPA (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH8, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, supplemented with ROCHE Complete protease inhibitor tablets (no EDTA), mixed gently by pipetting, and incubated on ice for 10 mins to achieve complete lysis. Extracted chromatin was then sonicated using Bioruptor (10 cycles of 30' on and 30', paused on ice for 5mins, repeated 10 cycles of 30' on and 30'). Sonicated material was then spun down at 20000g, at 40C for 15 mins. Supernatant was collected in fresh tubes and chromatin was quantified using Qubit high sensitivity DNA kit. 10µg of chromatin was used for each ChIP reactions, in a total reaction volume of 1ml, adjusted with RIPA. For each sample, 30 µl of Protein A/G beads preconjugated to 2 µg antibody (by incubation of antibody with washed beads resuspended in 500µl RIPA for 3h) was added to the reaction and chromatin was capture by overnight incubation at 40C, on a rotating platform. The next day, captured chromatin was washed successively as follows: once in low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), twice in high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), twice in lithium chloride wash buffer (250mM LiCl, 1% NP-40, 1% Sodium Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), and twice in 1X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Bound chromatin was then eluted in 200 µl freshly prepared direct elution buffer (10mM Tris-HCl pH8, 0.3M NaCl, 5mM EDTA pH8, 0.5% SDS) supplemented with 2 µl RNase A (10 mg/ml), by incubating on a thermomixer at 650C, at 800 rpm, overnight. The next day, the beads were discarded, and the supernatant was incubated with 3 µl of Proteinase K (20mg/ml) at 550C, 1200rpm, for 2h to reverse crosslinks. DNA was using the Zymo DNA Clean and Concentrator kit. For each sample, double stranded DNA (10µl), Template Preparation D Buffer (2µl), and Template Preparation D Enzyme (1µl) were combined and End Repair and A-tailing was performed in a Thermocycler with a heated lid (22ºC, 25 min; 55ºC, 20 min). Library Synthesis D Buffer (1µl) and Library Synthesis D Enzyme (1µl) were subsequently added, and library synthesis was performed (22ºC, 40min). Immediately after, Library Amplification D Buffer (25µl), Library Amplification D Enzyme (1µl), Nuclease-free water (4µl), and a unique Illumina-compatible indexed primer (5µl) were added. Library amplification was performed using the following cycling conditions: 72ºC for 3 min; 85ºC for 2 min; 98ºC for 2 min (denaturation); 4 cycles of 98ºC for 20 s, 67ºC for 20 s, 72ºC for 10 s (addition of indexes); 14 cycles of 98ºC for 20 s, 72ºC for 10 s (library amplification). Post-PCR clean-up was performed on amplified libraries with a SPRIselect bead 0.6X left/1x right double size selection then washed twice gently in 80% ethanol and eluted in 10-12µl 10 mM Tris pH 8.0. PE50