GSM3690890: Input DNA; Mus musculus; ChIP-Seq
Rib chondrocytes were manually dissected from ribs of P1 neonatal wild-type ICR mice to include proliferative and prehypertrophic regions and to exclude mature hypertrophic regions.Superficial soft tissue was removed, and ribs were washed in DMEM. Segmented ribs were initially digested in 0.05% Trypsin/EDTA (T4174; Sigma-Aldrich) for 30 minutes at 37 °C, and then in 0.3% Collagenase D (1088858; Roche) for 45 minutes at 37 °C.Rib chondrocytes isolated from newborn wild-type ICR mice were immediately cross-linked with 1% formaldehyde for 10 minutes at room temperature. The crosslinking was quenched by addition of 2.5 M Glycine at the final concentration of 130 mM. Twenty million cells were used for a ChIP reaction. After washing with phosphate buffered saline (PBS) twice, cells were lysed in Lysis buffer (50 mM HEPES-KOH pH 7.5,140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% IGEPAL CA-630, 0.25% Triton-X 100) at 4 ℃ with agitation for 10 minutes, and then washed with Buffer 2 (10 mM Tris-HCl pH8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 10 minutes at room temperature. Chromatin was sonicated in Buffer 3 (10 mM Tris-HCl pH8.0, 1 mM EDTA, 0.5 mM EGTA) by 10 sessions of 30 pulses (1 sec on and 1 sec off) at 50% amplitude using the Branson Advanced-Digital sonifier 250 (Branson Ultrasonics Corporation, Danbury, CT) in order to obtain 100 – 600 bp of DNA fragments. Lysate was cleared by centrifugation and addition of 20% N-Lauroylsarcosine Sodium Salt (L9150; Sigma-Aldrich, St. Louis, MO) at the final concentration of 0.5%. One hundred microlitters of Dynabeads M-280 Sheep anti-Rabbit IgG (112-03D; Life Technologies, Carlsbad, CA) was combined with 10 μg of anti-Sox6 antibodies (ab30455; Abcam, Cambridge, UK). The beads/antibody complex was incubated with the lysate overnight at 4 ℃. Beads were washed 5 times with RIPA buffer (50 mM HEPES-KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 0.7% Sodium Deoxycolate, 1% IGEPAL CA-630) and once with TE buffer containing 50 mM NaCl. DNA was eluted with Elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1% SDS) at 65 ℃ for 15 minutes, and the eluate was incubated overnight at 65 ℃ for reverse crosslinking. After the eluate was treated with RNase (R4642; Sigma-Aldrich) and proteinase K (25530015; Invitrogen), DNA was purified by MinElute PCR Purification kit (28004; Qiagen). Libraries were constructed with ThruPLEX DNA-seq Kit (R400429; Rubicon Genomics, Ann Arbor, MI) according to manufacturer's instructions.