GSM4711144: H3K27AC Old Sample 2; Mus musculus; OTHER
For CUT&RUN libraries 300K cells / sample were washed twice with 1.5ml Wash Buffer (20mM MHEPES, 150mM NaCl, 0,5mM Spermidine) and incubated with 200ul activated Concavalin A beads for 10minutes at RT. Cell-bead suspension was resuspended in 100ul antibody buffer (20mM MHEPES, 150mM NaCl, 0,5mM Spermidine, 0,05% Digitonin, 2mM EDTA) which was then divided into two 50ul tubes, and in each one of those 1ul of H3K27ac or H3K27me3 antibodies was added (antibody dilution 1:50 / antibody incubation O/N at 4C). Cell-bead suspension was then washed twice with Digitonin Buffer (20mM MHEPES, 150mM NaCl, 0,5mM Spermidine, 0,05% Digitonin) and 2.5 ul of EpiCypher CUTANA pAG-MNase were added in 50ul Digitonin Buffer, for 10 minutes. After washing samples twice with Digitonin buffer, 3ul of 100mm CaCl2 were added in 150ul of Digitoning buffer. Samples were incubated for 2hours at 4C. To stop the reaction 100ul of the STOP buffer (340mM NaCl, 20mm EDTA, 4mM EGTA, 0,02%Digitonin, 50ug/ml RNase A, 50ug/ml Glycogen, yeast genomic DNA) were added in each tube. Samples were incubated at 37C for 10minutes at 500rpm and after centrifugation, liquid was collected, DNA was purified using Zymo DNA clean concentrator kit and DNA was eluted in 10ul elution buffer. For library construction we followed a modified Ford et al., protocol (https://bmcresnotes.biomedcentral.com/articles/10.1186/1756-0500-7-312). In brief, we first blunted DNA ends by incubating ChIP-ed DNA for 30 minutes at 20C with T4 DNA ligase buffer (without ATP), 5 µl ATP 10 mM, 2 µl dNTPs 10 mM, 0,5 µl End-Repair-Enzyme-Mix(5µl T4 DNA Polymerase,1µl Klenow fragment, 5µl T4 PNK) in 17.5ul H2O. DNA was then cleaned-up using Ampure XP beads and was next subjected to poly-A addition at the 3'ends of DNA fragments. For addition of "A" bases, 2 µl NEB buffer2 10X, 1 µl dATP 4mM and 0,5 µl Klenow 3' to 5' exo minus 5 U/µl were added to each sample. Samples were incubated for 30 minutes at 37°C and 5 minutes at 70°, for inactivation. Next, adapters were ligated to each ChIP - sample by adding 27,25 µl 2X Quick ligase buffer, 2,5 µl annealed NEXTflex adapters, 3,75 µl H2O and 1 µl Quick Ligase (2000 U/µl NEB). Ligation reaction was incubated 15 minutes at RT and 15 minutes at 30°C and was then stopped by addition of 5,5 µl EDTA 0,5M pH 8.0. DNA samples were cleaned up by Ampure XP beads and libraries were amplified by PCR after addition of 1 µl PCR primer Mix and 20 µl HiFi HotStart 2X Kapa Mixm, as following: 45 seconds at 98°C/ 15 seconds at 98°C - 30 seconds at 63°C - 30 seconds at 72°C (12x) / 60 seconds at 72°C. DNA was cleaned up by Ampure XP beads and purified using Zymo DNA concentrator kit. Quantity of the libraries was assessed by measuring DNA concentration in Qubit library quality was assessed by running samples on the TapeStation.
GEO Accession
GSM4711144