GSM6935180: Tcf1_CUT&RUN_KO; Mus musculus; OTHER
Following sorting, CUT&RUN was performed on 350,000 cells/sample using the ChIC/CUT&RUN Kit (EpiCypher #14-1048) and anti-Tcf1 antibody (clone C46C7, Cell Signaling Technology Cat. No. 2206). The procedure followed the manufacturer's protocol with the following modifications. No spike-in controls were used, 1.5 μl anti-Tcf1 antibody/sample was used. pAG-MNase binding was performed for 30 min at 4C while rotating. MNase digestion was performed for 40 min at 4C while rotating. eBioscience Perm Buffer (Invitrogen/Thermo Fisher Scientific Cat. No. 00-8333) was used at 1x concentration (0.1% saponin) supplemented with Spermidine and cOmplete™EDTA-free Protease Inhibitor (Roche Cat. No. 11836170001) during the incubation step with pAG-MNase. 0.5x eBioscience Perm Buffer was used during the wash step post-pAG-MNase and at later steps of the protocol. Sequencing libraries were prepared with the NEBNext Ultra II Library Prep Kit (New England Biolabs Cat. No. E7645) and the Magnetic SPRI beads (Beckman Coulter Cat. No. B23317) for purification. PCR amplification conditions were: 14 cycles of DNA melting at 98˚C for 15 seconds, annealing plus extension at 60˚C for 10 seconds, followed by final extension at 72˚C for 1 minute. Libraries were quality-checked and quantified by Bioanalyzer (Agilent) and Qubit (ThermoFisher) and sequenced as PE150 on Illumina NovaSeq 6000.