GSM8030872: ChIPseq_Tead2_AviTag_knock_in_2iL_ESCs_input; Mus musculus; ChIP-Seq
For ChIP, cells stably expressed biotin-alone or biotin-TEAD2 were expanded and crosslinked with 1% formaldehyde. Crosslinked cells were sonicated and diluted tenfold with ChIP dilution buffer, and then incubated with M280 streptavidin dynabeads at 4°C overnight. Streptavidin dynabeads-bound DNA was subsequently washed twice with wash buffer 1 (2% SDS), once with wash buffer 2 (50 mM HEPES (pH 7.5), 1 mM EDTA, 500 mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100), once with wash buffer 3 (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and then twice with TE wash buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). ChIPed DNA was reverse-crosslinked and purified for DNA library construction followed by sequencing. For CUT&Tag, 100,000 cells were harvested and washed twice with 200 µL of wash buffer. 10 µL concanavalin A beads (Bangs Laboratories) were added per sample and incubated at room temperature for 15 min. Then, 100 µL of dig-wash buffer containing 2 mM EDTA and 1 µg of primary antibody were added. The primary antibody incubation was performed on a rotating platform at 4°C overnight. Two hundred microliters of dig-wash buffer were added to remove the unbound antibodies. Then, the reaction was incubated with pAG-Tn5 (homemade) at 4°C for 2 hr. Two hundred microliters of dig-med buffer were added to remove unbound pAG-Tn5 protein. Next, the cells were resuspended in 100 µL of tagmentation buffer and incubated at 37°C for 1 hr. To stop tagmentation, 2.25 µL of 0.5 M EDTA, 2.75 µL of 10% SDS and 0.5 µL of 20 mg/mL proteinase K were added and incubated at 55°C for 30 min and then at 70°C for 30 min to inactivate proteinase K. Then, DNA was extracted. The antibodies included H3K27ac (Active Motif, 39133), H3K4me1 (Active Motif, 39297). ChIP-seq libraries were constructed using the VAHTSTM Universal DNA Library Prep Kit for Illumina® V2 (Vazyme Biotech). After PCR library amplification, size selection of adaptor-ligated DNA was performed using Agencourt AMPure XP Beads (Beckman Coulter). The libraries were sequenced on the Illumina NovaSeq or Hiseq X-ten platform (Annoroad Gene Technology Co., Ltd.).  To generate the CUT&Tag libraries, 21 µL DNA were mixed with 2 µL of a universal i5 and a uniquely barcoded i7 primer (Buenrostro et al, 2015a) using a different barcode per sample. A volume of 25 µL of NEBNext high-fidelity 2 × PCR master mix (NEB) was added and mixed. The sample was placed in a thermocycler with a heated lid using the following cycling conditions: 72°C for 5 min (gap filling); 98°C for 30 s; 14 cycles of 98°C for 10 s and 63°C for 30 s; final extension at 72°C for 1 min; and hold at 8°C. Post-PCR clean-up was performed by adding 1 × volume of Ampure XP beads (Beckman Counter), and the libraries were incubated with beads at room temperature for 15 min, washed twice gently with 80% ethanol, and eluted in 25 µL of 10 mM Tris (pH 8.0). The libraries were sequenced on Illumina NovaSeq (Annoroad Gene Technology Co., Ltd.).