title: GSM8728357: Abcam-ab4729, 1/100, 15 PCR, SDS, replicate 1 [H3K27ac]; Homo sapiens; OTHER
library_name: GSM8728357
library_construction_protocol: Bench top CUT&Tag was performed as previously described (https://www.protocols.io/view/bench-top-cut-amp-tag-bcuhiwt6) using 500,000 exponentially growing K562 cells per experiment. Following tagmentation, either SDS- or column- based DNA extraction was conducted. SDS-based DNA extraction: PCR tubes were placed on a magnetic rack and solution allowed to clear. Supernatant was removed and beads resuspended in 50 μL TAPS Buffer (10 mM TAPS pH 8.5, 0.2 mM EDTA) at RT. Tubes were returned to a magnetic rack and supernatant removed. 5 μL SDS Release Buffer (10 mM TAPS pH 8.5, 0.1% SDS) was added to each tube at RT. After a quick spin, samples were incubated at 58°C for 1 hour. Next, 15 μL SDS Quench Buffer (0.67% Triton-X 100 in Molecular grade H2O) was added at RT. Column-based DNA extraction: 1.68 μL 0.5M EDTA, 0.5 μL 10% SDS, 0.44 μL 10 mg/mL Proteinase K were added to each 50uL sample. Samples were incubated at 55°C for 1 hour and spun quickly. Sample tubes were then placed on a magnetic rack and solutions allowed to clear. Supernatant was transferred to a new microcentrifuge tube and processed according to the ChIP DNA Clean & Concentrator (Zymo Research D5205) protocol. For library amplification in PCR tube format, 21 μL DNA was combined with 2 μL of universal i5 and uniquely barcoded i7 primer where a different barcode was used for each sample that was intended to be pooled together. 25 μL NEBNext HiFi 2× PCR Master mix was added, then the sample was gently mixed through and spun down. The sample was placed in a Thermocycler with heated lid following these conditions: 72°C for 5 min (gap filling); 98°C for 30 s; 11-15 cycles of 98°C for 10 s and 63°C for 30 s; final extension at 72°C for 1 min; and hold at 4°C. Following PCR, bead cleanup was conducted by addition of 1.1x Ampure XP beads (Beckman Coulter). Library and beads were mixed thoroughly, then spun down and incubated at RT for 10-15 min. Beads were gently washed twice with freshly prepared 80% ethanol using a magnetic rack, then the library was eluted with 20-30 μL 10 mM Tris-HCl pH 8.0 at RT.
identifiers.namespace: GEO
identifiers.id: GSM8728357_r1