title: GSM4677952: 200716_WT_MEF_THAP1; Mus musculus; ChIP-Seq
library_construction_protocol: Twenty million MEFs were fixed in fresh media by adding 37% formaldehyde (F1635, Sigma) to attain a final concentration of 1% and incubated at 37°C for 10 min. Fixation was quenched by addition of 1 M glycine (Sigma) in PBS at a final concentration of 125 mM. Cells were washed twice with cold PBS and pellets were snap frozen in dry ice and stored at -80°C. Fixed cell pellets were thawed on ice and resuspended in 2 ml of cold RIPA buffer (10 mM TrisHCl pH 7.5, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, supplemented with proteinase inhibitor (Complete Mini EDTA free, Roche)). Sonication was performed using the Covaris S220 sonicator at duty cycle 20%, peak incident power 175, cycle/burst 200 for 30 cycles of 60 s sonication and 30 s of pause at 4°C. Chromatin was clarified by centrifugation at 21,000 g at 4°C for 10 min and precleared with 80 μl prewashed Dynabeads protein A (ThermoFisher) for 30 min at 4°C. 40 μl of prewashed Dynabeads protein A were incubated with 10 μg of antibody in 100 μl of PBS for 30 min at room temperature under continuous mixing, washed twice in PBS for 5 min and added to 1 ml of chromatin followed by overnight incubation at 4°C on a rotator. Beads were then collected in a magnetic separator (DynaMag-2 Invitrogen), washed twice with cold RIPA buffer, twice with RIPA buffer containing 0.3 M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (10 mM Tris pH 8.0, 1mM EDTA) plus 0.2% Triton X-100, and once with TE. Crosslinking was reversed by incubating the beads at 65°C for 4 hr in the presence of 0.3% SDS and 1 mg/ml of Proteinase K (Ambion). DNA was purified using Zymo ChIP DNA clean and concentrator kit (Zymo Research) and eluted in 20 μl. The entire ChIP DNA was used to prepare Illumina sequencing libraries. End-repair was performed in 75 μl of T4 ligase reaction buffer, 0.4 mM of dNTPs, 4 U of T4 DNA polymerase (NEB), 13.5 U of T4 Polynucleotide Kinase (NEB) and 1.5 U of Klenow fragment (NEB) at 24°C for 30 min in a ThermoMixer C at 400 rpm. End-repair reaction was cleaned using 2X Agencourt AMPure XP beads and eluted in 16.5 μl of EB that was used for A-tailing reaction in 20 μl of NEBNext dA-Tailing reaction buffer (NEB) with 7.5 U of Klenow fragment exo- (NEB) at 37°C for 30 min. The 20 μl of the A-tailing reaction was mixed with Quick Ligase buffer 2X (NEB), 3,000 U of Quick ligase and 5 nM of annealed adaptor (Illumina truncated adaptor) in a volume of 50 μl and incubated at 25°C for 20 min. Ligation was stopped by adding 50 mM of EDTA and cleaned with 1.8X Agencourt AM- Pure XP beads and eluted in 15 μl of EB that was used for PCR amplification in a 50 μl reaction with 1 μM primers TruSeq barcoded primer p5, TruSeq barcoded primer p7, and 2X Kapa HiFi HotStart Ready mix (Kapa Biosciences). The temperature settings during the PCR amplification were 45 s at 98°C followed by 14 cycles of 15 s at 98°C, 30 s at 63°C, 30 s at 72°C and a final 5 min extension at 72°C . PCR reactions were cleaned with Agencourt AMPure XP beads (Beckman Coulter), run on a 2% agarose gel and a smear of 200-500bp was cut and gel purified using QIAquick Gel Extraction Kit (QIAGEN). Library concentration was determined with KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems). Sequencing was performed on the Illumina Nextseq500 (75bp single-end reads).
attributes.tag: GEO Accession
attributes.value: GSM4677952