title: GSM8505823: TCF21 V3; Mus musculus; ChIP-Seq
library_construction_protocol: Cardiac fibroblasts were cross-linked for 10 minutes on ice in fixing solution (0.88% formaldehyde, 0.1 M sodium chloride (NaCl), 1 mM EDTA, 0.5 mM EGTA and 50 mM Hepes-KOH (pH 8.0)). The cross-linking reaction was quenched for 5 minutes at room temperature by adding glycine directly to the cell culture plates to achieve a final concentration of 0.125 M. The cells were washed twice with PBS, scraped and pelleted at 2000 g in a centrifuge at 4 °C. The supernatant was removed and cell pellets were frozen at -80 °C until use. All buffers used in the following procedures apart from the sonication buffer were supplemented with protease inhibitors (PIA32955; Thermo Fisher Scientific) and 0.1 mM sodium butyrate before use. To obtain nuclei, frozen cell pellets were incubated in cell lysis buffer 1 (50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 and 0.25% Triton) for 10 minutes on ice. The lysed cells were Dounce homogenized using a piston A and then centrifuged at 2000 g for 5 minutes at 4 °C. After centrifugation, the pellet was incubated in cell lysis buffer 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and 10 mM Tris pH7.2) at room temperature for 10 minutes to separate the cytoplasm from the nuclei. The samples were Dounce homogenized again using a piston A and then centrifuged at 2000 g for 5 minutes at 4°C to pellet the cell nuclei. The supernatant was removed, and the nuclei were washed and resuspended with cold sonication buffer (TE with 0.1% sodium dodecyl sulfate (SDS)). The samples were sonicated on a Covaris S2 Sonicator (Covaris Inc.) for 5 minutes with SDS (peak power: 105, 10% duty factor and 200 cycles/burst) until DNA fragments were in the 400-800 base-pair range (settings: duty cycle 10%, peak incident power 105 Watts, 200 cycles per burst). Triton, glycerol, NaCl and sodium deoxycholate were added to the chromatin samples to achieve final concentrations of 1%, 5%, 150 mM and 0.1%, respectively. Samples were centrifuged at 14,000 g for 10 minutes at 4 °C and the supernatant containing the chromatin was collected and transferred to a new tube. The chromatin was pre-cleared with 15 µl Protein-A Dynabeads (10001D, Thermo Fisher Scientific) for 45 minutes under rotation at 4 °C, after which the beads were removed. ChIP for TCF21 was performed using an IP-Star Compact automation system (Diagenode) with 3 µg of chromatin solution, 3 µg of antibody (HPA013189; Sigma-Aldrich) and 12 µl of Protein-A Dynabeads. The Dynabeads were sequentially washed with buffer 1 (RIPA 150 mM NaCl: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate and 1% Triton X-100), buffer 2 (RIPA 250 mM NaCl), buffer 3 (50 mM Tris-HCl, pH 8.0, 2 mM EDTA and 0.2% N-lauroylsarcosine sodium salt) and buffer 4 (Tris-EDTA (TE) and 0.2% Triton X-100) for 15 minutes each. The immunoprecipitated chromatin was then subjected to ChIPmentation on the beads as previously described15. Tagmented DNA was incubated with proteinase K in elution buffer (TE with 250 mM NaCl and 0.3% SDS) to disrupt the crosslinks, for 4 hours at 65 °C. The DNA was purified using theQiagenMinEluteDNA kit. Library preparationand PCR amplificationoftheeluted DNA was prepared aspreviouslydescribed by Buenrostro, D. etal, 2013. Paired-end sequencing was obtained for pooled libraries using a NovaSeq 6000 instrument (Illumina, USA).