title: GSM5819711: JT_680_gata6RFPline1_0h_FLAG_chip1_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: 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. For the first round of ChIP, fixed pellets of undifferentiated cells and cells induced with Dox for 2 hours were thawed on ice for 10 mins. For each time point 6 replicates of 10 million cells each, were used. Nuclei were extracted and lysed from thawed cell pellets using the Covaris truChIP® Chromatin Shearing Kit, following recommendations for high cell numbers (1ml lysis buffer/ 20million cells). Lysed nuclei were sonicated in Covaris 1 ml AFA Fiber milliTUBE (Duration: 2100Secs, peak power-75W, duty factor 15%) to fragment chromatin to an average size of 200bp. Sonication chromatin was centrifuged at 20800g for 15mins to separate residual debris and the cleared lysate was transferred to a fresh pre-chilled tube. 25µl was aliquoted separately to confirm sonication efficiency and estimate chromatin concentration while the rest was frozen and stored at -80C. To the 25µl, 1 µl of RNase A (10 mg/mL) was added and incubated at 37C for 30 min after which 1 µl of Proteinase K (10 mg/mL) was added and chromatin was reverse cross-linked overnight at 65C in a PCR cycler with a heated lid. The following day, after performing 2X SPRI bead clean fragment size was confirmed by running 1ng on a tape station, and concentration was estimated using Qubit 1X dsDNA high sensitivity kit. Frozen chromatin was thawed and NaCl (final concentration of 150mM), Triton-x-100 (final concentration of 1%) and Na-deoxycholate (final concentration of 0.1%) were added. 10µg chromatin was used per replicate and a total of six replicates for each time point were subjected to immunoprecipitation with anti-FLAG M2 conjugated to protein A/G beads. 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). To elute FLAG-bound chromatin, beads were resuspended in 60µl of freshly made Direct Elution Buffer (10mM Tris-HCl pH8, 0.3M NaCl, 5mM EDTA pH8, 0.5% SDS) supplemented with 10mM DTT and incubated at 370C, 500rpm, 15mins. Eluted material was collected in a fresh tube and the process was repeated once more and pooled with the first elution. Finally, eluates from three replicates were pooled together to give two replicates per timepoint. 100µl was subjected to reverse crosslinking and library preparation to map FLAG-bound regions, and 100µl was added to 900µl RIPA and used for immunoprecipitation with anti-Nanog conjugated to protein A/G beads and the protocol described above was followed. 20 amplification cycles were used to prepare libraries. 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.