alias: OCT4-lin29-42 ChIP OSKM48h
title: OCT4-lin29-42 ChIP OSKM48h
design: human fibroblasts (HF176) were seeded at a density of 1.5x10*6 cells in 15 cm dishes and cultured in and cultured in GMEM supplemented with 10% FCS, 1X 13 non-essential amino acids, 1 mM Sodium Pyruvate, 1 mM glutamine and 0.1 mM 2-mercaptoethanol at 37C and 5% CO2. After 8 hours, the cells were transduced with concentrated OCT4 (WT or mutants) lentivirus, along with SOX2, c-MYC, KLF4 and rtTA2M2 concentrated lentiviruses at M.O.I of 5. After 16 hours of infection, the culture medium was replaced, and cells were cultured for 48 hours. OSKM protein expression was then induced adding fresh medium containing 1g/mL of doxycycline into one dish of each factor infection. Cells were cultured for 48 hours at 37C and 5% CO2 before collection. Cells were then washed 3X with PBS at RT. After final wash, 15mL of PBS/MgCl2 was added to each plate and 120L of fresh DSG (0.25M stock prepared by adding 613L DMSO to 50mg of DSG) were added to each plate to get a final concentration of 2mM. Plates were incubated at RT for 45min in a horizontal shaker with constant moving followed by 3X washes of PBS. After the last wash, 20 mL of PBS were added to each plate followed by adding 3mL of Formaldehyde crosslinking buffer (50mM HEPES-KOH, pH7.5, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 11% formaldehyde). Crosslinking was blocked by adding 1.65 mL 2.5M Glycine and incubating for 5 minutes at room temperature. Cells were harvested using a silicon scraper and centrifuged for 5 minutes at 1350 rcf at 4C. Cell pellets were then re-suspended in 10mL Lysis Buffer 1 (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40 Substitute, 0.25% Triton X100 and cOmplete Ultra Protease Inhibitor rotated for 10 minutes at 4C. Cells were disrupted using a tight 7mL dounce homogeniser with 80 strokes on ice. Nuclei were collected by centrifugation at 1350rcf for 5 minutes at 4C. The nuclei were washed in 10mL Lysis Buffer 2 (10mM Tris-HCl pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA and cOmplete Ultra Protease Inhibitor) for 10 minutes a rotator at room temperature. The nuclei were collected by centrifugation and resuspended in Lysis Buffer 3 (10mM Tris-HCl pH8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine and cOmplete Ultra Protease Inhibitor. Samples were sonicated for 140 min in 10 minutes intervals using a Covaris M220 sonicator. To solubilise chromatin following sonication, 100L of 10% Triton X-100 was added per 1mL of sonicated chromatin. Before ChIP, 50l of sonicated chromatin were collected to confirm optimum DNA fragmentation (~ 200 bp) was achieved by agarose gel electrophoresis. Three ChIPs were performed for each ChIP-seq sample (OCT4 WT and mutants).  Dyna G Magnetic Beads were transferred to a Prechilled LoBind tube and washed 3X with 1mL of Blocking buffer (0.5 BSA in PBS). Beads were resuspended in 800 l of Blocking buffer and incubated with 10g of OCT4 antibody per ChIP and incubated for 6 hr (minimum) on a rotating platform at 4C. Beads were washed 3X in 1mL of blocking solution and resuspended in 100l Lysis buffer 3.  Sonicated chromatin (40 g) were added to each ChIP and volume was top up to 900 l with Lysis buffer 3 buffer containing protease inhibitors. ChIPs were incubated overnight in a rotating platform at 4C.  To reduce background the ChIP reactions were transferred to fresh pre-chill LoBind tube. Beads were washed 4X with cold RIPA-wash buffer (50 mM Hepes-KOH pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) using magnetic rack. Beads were washed once with TE+NaCl (10mM Tris-HCl pH 8, 1mM EDTA pH 8 and 50mM NaCl), followed by a spin at 300 x g for 3 minutes at 4C and to remove any residual TE buffer. Bound chromatin fragments were eluted by addin 210 L elution buffer to the beads and incubated at 65C for 30 minutes shaking at 1200rpm. Tubes were centrifuged at 16,000 x g for 1 minute at room temperature and 200 L of supernatant was transferred to a new tube. In parallel, 50 L of sonicated chromatin from the INPUT after sonication was thawed before adding 150 L of ChIP elution buffer. ChIPs and input were reverse cross-linked by incubating at 65C overnight (shake 700rpm). An equal volume (200 L) of TE (10mM Tris-HCl pH8, 1mM EDTA) was added and incubated with 0.2mg/mL RNAse A for 2 hours at 37C. Proteins were then digested by incubating with 0.2mg/mL Proteinase K for 2 hours at 55C. To extract DNA, 400 L Phenol:chloroform:isoamyl alcohol were added to the samples to separate phases by spinning the sample at top speed for 5 min. The aqueous layer was transferred to a new tube containing 16 L of 5M NaCl (200 mM final concentration) and 1.5 L of 20 g/L glycogen (30 g total). DNA precipitation was performed by adding 0.8mL of cold 100% EtOH and incubated for 180 min at least at -20C. Tubes were centrifuged 20,000 x g for 10 minutes at 4C to pellet DNA. Supernatants were carefully decanted without disturbing the DNA pellet, which was further washed with 500 L of 80% EtOH and spining at 20,000 x g for 10 minutes at 4C. Supernatants were removed by pipetting and pellets were allowed to dry for 5 min. Each pellet was resuspended in 20 L MiniPrep Elution buffer (10mM Tris-HCl pH8.5, Qiagen) using a P20 pipette. DNA was quantified by Qubit 2.0 using the HS dsDNA quantification kit. DNA libraries were prepared using 20-50ng ChIP-DNA prepared by pooling equal volumes of 3X ChIP replicates for each OCT4 (WT and mutant) using the NEBNext Ultra II Library Preparation Kit with Dual Index Primers. Size selection for DNA fragments of approximately 200bp was carried out using SpeedBeads, which comprise magnetic carboxylate modified particles. PCR amplification during library preparation was limited such that samples underwent 10 cycles of PCR amplification. Libraries were quantified using a Qubit 2.0 device with a high sensitivity dsDNA kit and fragment size was determined using an Agilent 2200 Tapestation with D1000 HS reagents. The DNA libraries were then pooled by mixing an equal concentration of each ChIP DNA library. The concentration of the ChIP DNA library pool was determined to be 24.8 nM by an Agilent 2200 Tapestation with D1000 HS reagents. Library was submitted for sequencing using a NovaSeq S1 50PE (1 lane) platform to yield approximately 750M + 750M paired ends. Sequencing was performed by Edinburgh Genomics (Kings Buildings, The University of Edinburgh).
identifiers.namespace: SUB7272244
identifiers.id: OCT4-lin29-42 ChIP OSKM48h