title: GSM6721427: Cut&Tag data for Myc in teton MED30 expression Mia PaCa2 cells , +dox 1day; Homo sapiens; OTHER
library_construction_protocol: cells were seeded in 24-well plate at 30-50% confluency, and treated with indicated doxycycline concentration for two days to allow the induction of shRNAs or cDNA plasmid, and the attachment to the well's bottom. On the day of experiment, cells in culture media were lightly fixed (0.1 % formaldehyde for 2 minutes) for non-histone epitopes or without fixation for histone marks, and quenched glycine of >twice molar concentration of formaldehyde. Next, the cells were washed with PEX buffer (PBS, 2mM EDTA, 0.1% Triton X-100) for 5min thrice, in order to complete remove Mg2+ and permeabilize the cell. Then the cells were blocked by antibody buffer (0.1%BSA, 2mMEDTA) for 10-30min at room temperature, and subjected to primary antibody incubation overnight at 4°C. The antibodies were used as 1:1000 dilution in antibody buffer if not specified. The next day the primary antibody was removed and wash with wash buffer (20mMTrisCl pH7.4, 150mMNaCl, 0.05%Triton X-100) for 5min, and then incubated with secondary antibody 1:2000 diluted in wash buffer for 1h. The secondary antibody contains fluorophore (Alexa FluorTM) so the signal can be checked under microscope. After secondary antibody incubation, cells were washed three times with wash buffer, and then add adapter loaded pAG-Tn5 (purified in-house [3]) in Dig-300 buffer (20mMTrisCl pH7.4, 300mMNaCl, 0.05%Triton X-100) for 1h, followed by three time wash in Dig-300 buffer. Then cells were subjected to tagmentation buffer (10mMMgCl2 in Dig-300 buffer) at 37°C for 1-3h, and terminated and de-crosslinked by adding 7.5ul 0.5MEDTA, 8ul 10%SDS, 0.25ul 20mg/ml proteinase K per 150ul tagmentation buffer in one well and incubated at 65 °C for 30 min, and transferred into Eppendorf tube to avoid evaporation and continue de-crosslinking overnight, followed by DNA purification with DNA purification Kit (Qiagen). The DNA was eluted and utilized as template for PCR reaction with i7 and i5 dual-index for library construction and sequenced on the NovaSeq 6000 with 100 bp paired-end reads at the UCSD IGM Genomics Center.