title: GSM4801080: Uhrf_DKO0_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were washed once with PBS and fixed with 1% formaldehyde (methanol-free) for 10 minutes at room temperature with rotation. The formaldehyde was quenched with 125 mM glycine for five minutes at room temperature. Cells were spun at 500g for 10 minutes at 4°C and washed twice with ice-cold PBS supplemented with Protease Inhibitor cOmpleteTM. Subsequent work was performed on ice and with buffers cooled to 4°C. The pellet was lysed in L3B buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Sodium Deoxycholate (DOC), 0.5% N-lauroylsarcosine, 1x protease inhibitors) and sonicated in a 130 µL milliTUBE in a Covaris E220 for 7 min until most of the fragments were 200- 700 base pairs long (settings: duty cycle 5%, peak incident power 140 Watts, cycles per burst 200). Lysates were supplemented with 1% Triton-X-100 and centrifuged at full speed for five minutes at 4°C, the supernatant containing the sonicated chromatin was transferred to a new tube. In parallel, Protein G magnetic beads (Invitrogen) were blocked and conjugated to an antibody by washing them twice in PBS with 0.5% BSA and resuspended in 200 μl of PBS with 0.5% BSA per IP. Anti-Flag (1 μg, Millipore/Sigma F1804) was added and bound to the beads by rotating >1 hour at room temperature. Blocked antibody conjugated magnetic beads were added to the tube containing the chromatin and incubated over night at 4°C. The beads were then washed twice with each of the following: TFWBI (20 mM Tris-HCl/pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA), TF-WBIII (250 mM LiCl, 1% Triton X-100, 0.7% DOC, 10 mM Tris-HCl/pH 8, 1 mM EDTA), and 10 mM Tris-HCl pH8. Beads were resuspended in 24 μl of tagmentation buffer and 1 μl of Tn5 transposase (Illumina 15027866, 15027865) and then incubated at 37ºC for five minutes in a thermocycler. Tagmentation reactions were removed and beads were washed twice with WBI and TET (0.2% Tween-20, 10 mM Tris-HCl/pH 8.0, 1 mM EDTA) (twice). Beads were then incubated with 70 μl elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0) containing 2 μl of Proteinase K (NEB) for 1 hour at 55°C and 8 hours at 65°C to reverse crosslink, and supernatant was transferred to a new tube. Another 30 μl of elution buffer was added to the beads and incubated with another 1 μl of Proteinase K for 1 hour at 55°C, eluates were combined. Finally, DNA was purified with AMPure XP beads (sample-to-beads ratio 1:2). Relative quantitation was performed using SYBR-green as in (Schmidl et al, 2015; PMID:26280331) using 2 μl of DNA. Libraries were amplified according to the Cq values obtained in the previous step (12 cycles were used), purified using AMPure XP beads and eluted in 15 μl of water. 
attributes.tag: GEO Accession
attributes.value: GSM4801080