title: GSM4059018: GPA_Input_Rep2; Mus musculus; ChIP-Seq
library_construction_protocol: 6 days old EBs treated with 2 µg/ml doxycycline at day 4 were dissociated using 0.25% trypsin-EDTA (Invitrogen) in PBS and around 15x10^6 to 20x10^6 of cells were aliquoted into 15ml falcon tubes for fixation. Cells in each aliquot were fixed at room temperature for 12 minutes on 1% of formaldehyde solution (37% formaldehyde, Sigma-Aldrich). Formaldehyde was quenched with glycine (final 0.125 M) for 5 minutes at room temperature. Samples where then washed once with cold PBS containing protease inhibitors (cOmplete protease inhibitor cocktail, Roche). After, cells were centrifuged at 500 g and pellets were snap frozen on dry ice followed by storage at -80C until further use. The pellet for each sample (~20x10^6 of cells) was thawed and nuclei were isolated by re-suspension in lysis buffer (5 mM Pipes pH 8.0, 85 mM KCl, 1% NP40) with fresh protease inhibitors (cOmplete protease inhibitor cocktail, Roche) for 20 minutes on ice followed by brief vortexing and centrifugation at 500 g for 10 minutes at 4C. The nuclear pellet was re-suspended in 300 ul IP buffer (0.5% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, fresh protease inhibitors) and sheared by sonication (BioRuptor Pico, Diagenode) down to ~200-300 bp fragments using a total of 5-14 cycles of: 10 pulses of sonication on the ''high'' setting, each followed by 30 s off. Samples were then diluted in IP buffer without SDS, then 10 mg of the following antibodies was added for overnight incubation at 4C on a rotator: Monoclonal anti-flag M2 antibody (F1804, Sigma); Myc tag antibody-ChIP Grade (ab9132, Abcam); Anti-Gfi1 antibody (ab21061, Abcam). Dynabeads-ProteinG (Life Technologies) were blocked for 1-hour in 0.5% BSA in IP buffer with protease inhibitors, prior to incubation with the sonicated antibody bound chromatin suspensions. Bead-chromatin complexes were then serially washed for 5 minutes each with the following solutions: IP buffer (150 mM NaCl), followed by IP buffer with high salt concentration (500 mM NaCl), then 1 wash with washing-buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 0.5% NP40, 0.5% Na-Deoxycholate, 1 mM EDTA), followed by 2 washes with TE (10 mM Tris, 1 mM EDTA). The DNA-protein complex was then eluted from beads by incubation with 100 ml of elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0) for 30 minutes at 65C, vortexing every 10 minutes, followed by magnet extraction of beads. The beads were re-washed with 150 ml TE with 1% SDS, magnet extracted, and the TE with remaining DNA solution was added to the eluted samples, followed by 65C overnight incubation to reverse crosslink the DNA-protein complexes. The dissociated DNA and protein solution was then treated with 4U of proteinase K (NEB) at 37C for 2 hours. DNA was isolated with SeraMag beads, using 450 ml SeraMag bead solution and 225 ml of 30% PEG in 1.25 M NaCl. After bead purification, DNA was re-suspended in 30 µl of ddH2O (DNase/RNase free) and for each ChIP sample, DNA concentration was estimated using Qubit fluorometric quantification (Invitrogen) before being stored at 80C. 8-10 ng of ChIP DNA was used for each sample. DNA ends were blunted by treatment with 5 µl T4 DNA ligase buffer (NEB), 2 µL 10 mM dNTP's, 0.5 µl end repair mix (0.72 U T4 DNA polymerase, 0.24 U Klenow Fragment, 2.4 U T4 DNA Polynucleotide Kinase), up to 50 µl with ddH2O (DNase/RNase free). Samples were incubated for 30 minutes at 20C, and then purified with addition of 50ul SeraMag bead solution and 50 µl 30% PEG solution (in 1.25 M NaCl). DNA was eluted in 16.5 ul of TE/10 (10 mM TrisCl pH 8.0, 0.1 mM EDTA). DNA was then A-tailed at the 3' ends by treating the eluate with 2 µl 10X NEB Buffer 2, 1 µl 4 mM dATP, 0.5 µl Klenow 3' to 5' exonuclease minus (NEB) in a total volume of 20 µl. Reaction was incubated at 37C for 30 minutes. Before adapter ligation, TruSeq adapters (Illumina) were first annealed by re-suspending each adapter at 100 mM (in 10 mM Tris-HCl pH7.8, 0.1 mM EDTA pH 8.0, 50 mM NaCl) and mixing them at a 1:1 ratio. Adaptor annealing occurred using a program of 2 min at 95C, 70 cycles of 30 s (95C decreasing by 1C each cycle). Annealed adaptors were diluted 1:200 (0.25 mM final) and then 1 µl was used for ligation reaction containing 20 µl of the A-tailed DNA, 1.5 µl Quick Ligase (2,000 U/ml NEB) and 2.5 µl of H2O. The reaction was incubated 20 minutes at room temperature and stopped by addition of 5 µL of 0.5 M EDTA pH 8.0. Next, DNA was purified using 50 µl SeraMag bead mix and 50 µl 30% PEG (in 1.25 M NaCl) and eluted in 15.5 µl of TE/10. Next, the adapter ligated DNA was amplified using the following protocol: 15 µl adaptor-ligated DNA, 1 µl TruSeq primer cocktail (0.25 mM), 15 µl 2X Kapa HiFi HotStart ready mix. The Libraries were amplified with the following protocol: 45 s at 98C, 5 cycles of (15 s at 98C, 30 s at 63C, 30 s at 72C), 1 minute of 72C, hold at 4C. After pre-amplification of the library, a size selection step was performed to obtain DNA fragments between 300 and 500 bp using the following procedure: 0.9X beads were added to ChIP DNA After 15 minutes incubation and 10 minutes magnet extraction, the supernatant was transferred to new tube and beads discarded. Then 0.2X beads were added to the solution, incubated 15 minutes followed by 10 minutes magnet extraction and beads re-suspension in 11.5 µl TE/10. 11 µl of pre-amplified and size selected DNA was further amplified with 1 µl TruSeq primer cocktail, 20 µl 2X Kapa HiFi HotStart ready mix, and 8 µl H2O using a PCR program of 45 s at 98C, 9-11 cycles of (15 s 98C, 30 s 63C, 30 s 72C), 1-minute 72C. After amplification, the DNA was isolated using SeraMag bead purification. Libraries were assessed using Agilent High Sensitivity D1000 ScreenTape System on an Agilent 2200 TapeStation. Finally, libraries were pooled at equal concentration and sequenced on the Illumina HiSeq 4000 platform.
attributes.tag: GEO Accession
attributes.value: GSM4059018