title: GSM3228218: 24plusCu_IP_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Differentiating (24 h) primary myoblasts were crosslinked with 1% Formaldehyde and incubated for 10 min at room temperature in an orbital shaker. To inactivate the formaldehyde, 1 ml of 1M glycine was added and cells were incubated for 5 minutes in an orbital shaker at room temperature. Cells were washed 3 times with 10 ml of ice cold PBS supplemented with Complete Protease Inhibitor (Roche). Crosslinked myoblasts were resuspended in 1 ml of ice cold PBS supplemented with Complete. The cell suspension was transferred to a 1.5 ml microcentrifuge tube and centrifuged for 5 minutes at 5,000 x g at 4 ºC. The PBS was removed and the cell pellet was resuspended in 200 µl of ice cold SDS lysis buffer (50 mM Tris pH 8; 10 mM EDTA, 1% SDS) for 10 min. Proliferating myoblasts were sonicated three times for 5 min 30” by 30” at mild intensity for and five times for nascent myotubes using a Bioruptor UCD-200 (Diagenode). The samples were diluted to a final volume of 1 ml in IP buffer (16 mM Tris pH 8.1; 1.2 mM EDTA; 0.01% SDS; 1.1% Triton X100; 167 mM NaCl). ChIP was performed using a rabbit anti-MTF1 and a rabbit IgG antibodies (Santa Cruz biotechnologies). Samples were incubated for 2 h at 4 ºC in agitation and 80 µl of Magna ChIP protein A+G Magnetic Beads (Millipore, Sigma) were added to each sample and incubated overnight in agitation at 4 C. The samples were then placed in a magnetic rack and washed using 1 ml of the wash buffer sequence A-D (Buffer A: 20 mM Tris pH 8.1, 2 mM EDTA, 0.1% SDS, 1% Triton-X100, 167 NaCl, Buffer B: 20 mM Tris pH 8.1, 2 mM EDTA, 0.1% SDS, 1% Triton-X100, 500 NaCl; Buffer C: 10 mM Tris pH 8.1, 1 mM EDTA, 1% NP40, 1% Sodium deoxicholate, 0.25 M LiCl2; Buffer D: 10 mM Tris pH 8.1, 1 mM EDTA). Immune complexes were eluted in 100 µl of buffer containing 0.1 M NaHCO3, 1% SDS, 1 µg/µl proteinase K. Samples were then reverse-crosslinked using 20 µl of 5M NaCl and incubated overnight at 65 ºC. The reverse crosslinked DNA was purified using the ChIP DNA clean concentrator, following the manufacturer's instructions (Zymo Research). Samples were end-repaired, A-tailed, and adaptor-ligated using barcoded inline adaptors according to the manufacturer's instructions (Illumina). DNA was purified over a Zymo Research PCR purification column between each enzymatic reaction (Zymo Research). DNA was PCR amplified with KAPA HiFi polymerase using 16 cycles of PCR. Each library was size-selected for 200-300 bp fragments on a 1.5% agarose gel and the library concentrations were determined using a QuBit 3.0 Fluorometer (Thermo Scientific). Libraries were sequenced on an Illumina HiSeq2000 using single-end 50bp sequencing at the UMass Medical School deep sequencing core facility Libraries of ChIP-enriched DNA were prepared following Illumina strategy
attributes.tag: GEO Accession
attributes.value: GSM3228218