GSM6031781: PRIMARYCHON_CNR_7365_PBS_0180; Homo sapiens; OTHER
Primary chondrocytes were washed with 1X PBS and treated with trypsin-EDTA (0.25%) for 5 minutes. Trypsin was quenched and cells were pelleted at 4°C for 5 minutes at 1000g. Cells were resuspended in 1mL plain DMEM per million cells and crosslinked in 1% formaldehyde for 10 min with rotation before quenching in a final concentration of 125mM glycine for 5 min with rotation. Cells were pelleted by spinning at 1000g for 5 min at 4°C. Each 2 million cell pellet was washed in 1mL cold PBS prior to flash freezing in liquid nitrogen. Thawed pellets were resuspended in 1mL ice-cold nuclei isolation buffer (NE1 buffer; 20mM HEPES pH 7.5, 10mM KCl, 1mM MgCl2, 1mM DTT, 0.1% Triton X-100, 1X CPI added fresh) and incubated for 10 min at 4°C with rotation. Nuclear pellet was collected by centrifugation at 1000g for 5 min at 4°C, then resuspended in 1mL of ice-cold wash buffer (WB; 20mM HEPES pH 7.5, 0.2% Tween-20, 150mM NaCl, 150mM BSA, 0.5mM Spermidine, 10mM Na-Butyrate, 1X CPI added fresh). 10uL concanavalin A lectin beads washed and resuspended in binding buffer (BB; 20mM HEPES pH 7.5, 10mM KCl, 1mM CaCl2, 1mM MnCl2) were added to each sample and incubated for 10 min at RT. Beads were reclaimed and resuspended in 50uL antibody buffer (AbB; WB supplemented with 0.1% Triton X-100 and 2mM EDTA). 0.01ug/uL H3K27ac antibody in AbB was added to each sample and samples were incubated overnight at 4°C with mixing at 1000xRPM. Beads were reclaimed and washed with 1mL triton wash buffer (TwB; WB supplemented with 0.1% Triton X-100) without mixing. Beads were reclaimed and resuspended in 50uL AbB. 2.5uL CUTANA pAG-MNase (Epicypher, #15-1016) was added and samples were incubated for 1 h at 4°C on a metal block. Beads were reclaimed and washed twice with 1mL TwB before resuspension in 100uL TwB. To digest chromatin, 2uL 100mM CaCl2 was added and samples were incubated for 30 min at 4°C on a metal block. Digestion was halted by the addition of 100uL 2X STOP buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 0.1% Triton X-100, 50ug/mL RNAse A). Samples were incubated for 20 min at 37°C to release pA-MNase cleaved fragments from nuclei). Beads were placed on a magnet and the supernatant containing DNA fragments was transferred to a new tube. To reverse crosslinks, 2uL 10% SDS and 2uL 20mg/mL proteinase K were added to each sample and incubated for 1 h at 65°C. DNA was purified using the Zymo DNA Clean & Concentrator Kit according to manufacturer's protocols using 5 volumes of DNA binding buffer. DNA was eluted in 55uL water. Sequencing libraries were prepared from CUT&RUN fragments using KAPA HyperPrep with library amplification kit (no. KK8504) following the manufacturer's instructions. Post-ligation bead cleanup was performed with two rounds of 1.2X volumes of beads and DNA was eluted in a final volume of 25uL 10mM Tris-HCl pH 8. Library amplification was performed with 20uL of the adapter ligated DNA with 12 PCR cycles. One round post amplification cleanup was performed with 1.2X volumes of beads. The resulting libraries were then quantified by Qubit and Tapestation. A low depth sequence was performed first using the Miniseq sequencer system (Illumina) and analyzed using the Juicer pipeline to assess quality control. The resulting libraries underwent paired-end 2x150bp sequencing on an Illumina NextSeq sequencer.