title: GSM6051631: EBF1-dTAG_C, 6h, input; Mus musculus; ChIP-Seq
library_construction_protocol: EBF1 ChIP was performed according to Boller et al. 2016 protocol with minor changes. Cells were crosslinked with formaldehyde. 4 µg rabbit a-EBF1 (1C) rabbit antibody per sample was used. Cells were harvested and washed with PBS. Cell pellet was resuspended in PBS with 2% FCS (4 mln cells per 1 ml buffer). Fresh formaldehyde mix (50mM HEPES-KOH pH8.0, 100mM NaCl, 0.5mM EGTA, 1mM EDTA, 11% (v/v) formaldehyde) was added to the cell suspension to a final concentration of 1% FA. Tubes were incubated on rotator for 10 min at RT. Quenching was performed by adding 2 M Glycine to the final concentration 0.2 M. Cell pellet was resuspended in Lysis buffer (50mM Tris-HCl pH 8.0, 1% (w/v) SDS, 10mM EDTA; PIM) (4 mln cells per 100 μl buffer). Chromatin was sheared the chromatin with a Bioruptor (20-25 cycles, output level "High", 30 sec ON, 30 sec OFF, 4°C). Fragment size and amount of chromatin was checked. 10-20 μg DNA was used for 1 ChIP reaction. Chromatin was mixed with 4 μg of EBF1 antibody or rabbit IgG and incubated on rotator ON at 4°C. Chromatin was incubated with 30 μl of Dynabeads Protein G (ThermoFisherScientific) suspension per sample on rotator for 4 hours at 4°C. Beads were washed 4 times with Wash buffer A (20mM Tris-HCl pH 8.0, 150mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 2mM EDTA); once with Final wash buffer A (20mM Tris-HCl pH 8.0, 500mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 2mM EDTA). Every wash was on rotator for 5 minutes at 4°C. Chromatin was eluted with 100 μl Elution buffer (10 mM Tris-HCl, pH 8.0; 0.5% SDS; 300 mM NaCl; 5 mM EDTA pH 8.0) by shaking vigorously at 65°C for 30 min. Samples were revese crosslinked, treated with RNase A and Proteinase K, and purified with QIAquick PCR Purification Kit. NEBNext Ultra II DNA Library Prep Kit for Illumina was used for library preparation according to the manufacturer's protocol.