GSM3291590: primed_Oct4_ChIP_2; Homo sapiens; ChIP-Seq
For chromatin immunoprecipitation, 2x10^7 H9 primed or naive hESC were harvested in 9 ml of medium and cross-linked by addition of 270 ul 37% Formaldehyde (Sigma, final concentration of 1%), for 10 min at room temperature under rotation. 1 ml of 1.25 M Glycine was added, cells were incubated on ice for 5 min and 3x washed with ice cold PBS. At this point, cross-linked cell pellets were snap-frozen and stored at -80ᴼC, or immediately processed for sonication. Prior to sonication, cells were resuspended in 1ml TE-I-NP40 (10mM TRIS-HCl pH 8, 1mM EDTA, 0.5% NP40, 1mM PMSF, 1x Protease inhibitor complex (PIC, Complete tablets, 04693116001, Roche)) incubated on ice for 5 min and centrifuged for 5 min at 2500 rpm at 4ᴼC in a refrigerated bench top centrifuge (Eppendorf). Supernatant was removed and nuclei were resuspended in 1 ml ice-cold lysis buffer (50mM TRIS-HCl pH 8, 10mM EDTA, 1% SDS, 1mM PMSF, 1x PIC) and transferred to a 15 ml Falcon tube for sonication, using a Diagenode Bioruptor Next Gen (40 cycles of 30” on, 30” off). After transfer to an Eppendorf tube and centrifugation for 10 min at 13200 rpm at 4ᴼC, chromatin solution was aliquoted and used for immunoprecipitation or snap-frozen and stored at -80ᴼC. A 20 µl sample was taken and served as a total input control. For immunoprecipitation, Protein Dynabeads G (10004D, Life Techology) were washed with PBS and incubated for 6 hours with 5 ug of antibody, at 4ᴼC on a rotating wheel. Antibodies used were: goat-anti-NANOG (AF1997, R&D Systems), rabbit-anti-OCT4 (AB19857, Abcam), rabbit-anti-H3K4me1 (AB8895, Abcam) and rabbit-anti-H3K27ac (AB4729, Abcam); as a control, respective IgG antibodies were used (rabbit-IgG: 10500C, Life Technology, goat-IgG: SC-2028, Santa Cruz Biotechnology). After washing with PBS, antibody-coupled beads were incubated with 200 ul chromatin solution, diluted to a final volume of 2 ml with dilution buffer (167mM NaCl, 16.7mM TRIS-HCl pH 8.1, 1.2mM EDTA, 0.01% SDS, 1.1% Triton-X100, 1mM PMSF, 1x PIC), overnight at 4ᴼC on a rotating wheel. Washing of beads was performed by incubation with ice-cold 1 ml of washing buffer, for 5 min, at 4ᴼC on a rotating wheel, followed by removal of supernatant using a magnetic stand, for each of the following: 2x with wash buffer 1 (10mM TRIS-HCl pH 7.6, 1mM EDTA, 0.1% SDS, 1% Triton-X100, 0.1% NaDeoxychloate), 2x with wash buffer 2 (10mM TRIS-HCl pH 7.6, 1mM EDTA, 0.1% SDS, 1% Triton-X100, 0.1% NaDeoxychloate, 150mM NaCl), 2x with wash buffer 3 (250mM LiCl, 0.5% NP40, 0.1% NaDeoxychloate), 1x with TE 1x with 0.2% TritonX-100 and 1x with TE 1x, after which beads were resuspended in 100ul TE1x. Immunoprecipitated chromatin and total input control were decross-linked, by addition of 3 ul of 10% SDS and 5 ul Proteinase K (20 ug/ul, Roche) and 10 ul RNAse A (50 ug/ul, Roche) to each tube and incubation overnight at 65°C on a shaking thermomixer block, 1400 rpm (Eppendorf). The next day, beads were briefly vortexed and supernatants were transferred to new tubes using the magnetic stand. 100ul of TE1x containing 500mM NaCl was added to the beads and briefly vortexed, after which the supernatant was added to the first fraction of collected supernatant. Following Phenol / chloroform extraction, DNA was precipitated using 1ul glycogen (20mg/ml), 1/10 vol NaOAc (3M) and 100% ice-cold Ethanol, at -20°C for 1 hour, followed by centrifugation at 13200 rpm for 1 hour at 4ᴼC. After a final wash with 70% ethanol, the DNA pellet was dried and resuspended in 50ul H2O. Concentration of ChIP DNA was determined by Qubit measurement following manufacturer's instructions and sonication was assessed by gel-electrophoresis of total input DNA (target fragment size between 200 and 600 bp). For ChIP-seq library generation, 10 ng of ChIP DNA was used as starting material. Using NEB Next ChIP-seq library preparation kit (E6200 or E6240, NEB), DNA was end-repaired, dA-tailed and adapter-ligated according to manufacturer's instructions. After adapter ligation and purification using AMPure-XP beads (0.8x, Beckman Coulter) and elution into 30ul of 0.1xTE, 25 ul of the reaction product was used for ChIP-seq library preparation, by PCR amplification with Illumina index primers (7335 and 7500, NEB) using the NEB Next Q Hot start high fidelity master mix (M0543S, NEB) according to manufactures instructions (cycle conditions: 98ᴼC 30 sec, (98ᴼC 10 sec, 65ᴼC 75 sec) x15, 65ᴼC 5 min, 4ᴼC hold). After an additional round of AMPureXP bead purification, DNA was eluted in 0.1xTE without further size selection. Quality and quantity of the prepared ChIP-seq libraries was assessed on an Agilent Tapestation.
GEO
GSM3291590