title: GSM9057431: ChIP_tbp_6h_rep3; Mus musculus; ChIP-Seq
library_name: GSM9057431
library_construction_protocol: ESCs were washed with PBS and fixed with ChIP cross-link GOLD reagent in PBS containing 1 mM MgCl2 for 30 minutes at room temperature. The cells were washed twice with PBS and fixed with 1% Formaldehyde for 10 minutes. Formaldehyde was quenched by the addition of Glycine to 125 mM. Fixed cells were scrapped and washed twice with chilled PBS, pelleted, and resuspended in NP Rinse 1 buffer (50 mM HEPES, pH 8.0, 140 mM NaCl, 1 mM EDTA, pH 8.0, 0.5 mM EGTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and incubated for 10 min on ice to isolate nuclei. The cells were pelleted at 1200g for 10 min and resuspended in NP1 Rince buffer 1 again and incubated for a further 10 min on ice and pelleted at 1200g.  The resulting nuclei were suspended in NP Rinse 2 buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, 200 mM NaCl) and incubated on ice for 10 min. The pellet was washed twice with shearing buffer (10 mM Tris-HCl, pH 8.0, 0.1% SDS, 1 mM EDTA, pH 8.0) without disturbing the pellet. Pellets were resuspended in shearing buffer with protease and phosphatase inhibitor cocktail and sonicated in a Covaris E220 sonicator for 20 min using settings 5% duty cycle, PIP 140 and 200 cycles burst, to generate DNA fragments between approximately 200 and 1,000 bp in length. Insoluble material was pelleted by centrifugation for 15 min at 16,000g. Chromatin was immunoprecipitated overnight at 4 °C with antibodies bound to ChIP-grade Protein A/G Magnetic beads in ChIP buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, 1 mM EDTA, pH 8.0, 1% Triton X-100, 0.1% DOC, 0.1% SDS). After incubation, bead slurry was washed 5 times with ChIP buffer, once with DOC buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% DOC, 1 mM EDTA pH 8.0) and once with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0). Chromatin was eluted with elution solution (0.1% NaHCO3, 1% SDS, and 1 mM EDTA) by incubating at 55 degrees for 15 min. After RNase A and Proteinase K digestion and de-crosslinking at 65 °C overnight, DNA was extracted with ChIP DNA Clean & Concentrator. In case of TAF1 and TBP ChIP-seq, human chromatin was used as a spike-in control. Experimental chromatin samples were mixed with a defined amount of HeLa cell chromatin, prepared using the same protocol as for the experimental ChIP-seq samples. A minimum of 2 ng of DNA was used for library preparation according to the NEBNext Ultra II DNA Library Prep Kit for Illumina. Purity and size distribution were analyzed on Tapestation 2200. The constructed libraries were pooled and subjected to single-end sequencing with 75 base pairs read length on a NextSeq 500 platform.