GSM7066484: Naïve CD8T RNAPII ChIA-PET; Mus musculus; ChIA-PET
Briefly, approximately 100-200 million cultured primary T cells were harvested and fixed with 45 mL of 1.5 mM Ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester) in DPBS buffer for 20 min at room temperature with 15 rpm rotating. A final concentration of 1% formaldehyde was added directly and followed by a rotation with 15 rpm at room temperature for 15 min. Before washing, quenched the crosslinked sample by adding a final concentration of 125 mM glycine at 15 rpm rotating for 10 min at room temperature. Collected all cell pellets with 1000 rcf for 5 min at 4℃ and washed the cell twice with 45 mL PBS before harvesting the cells. The dual crosslinked were aliquoted into 10 million cells each 1.5 mL LowBand tube for following library constraction or storing at a -80 ℃ freezer. The chromatin was obtained and sonicated to generate fragments with an average length of 1-3 kb. The monoclonal antibody against RNA Polymerase II pre-incubated protein G beads were used to enrich RNAPII-bound chromatin fragments by overnight rotating with 10 rpm at 4℃. The on beads chromatin DNA fragments were washed following 4 different cold buffers and rinsed in 100 μL TE-1% SDS solution for 1 hour at 65℃ with 1100 rpm rotation. The solution was added 150 μL Qiagen EB buffer for another 45 min decrosslink. Collected all supernatant, added 10 μL proteinase K, and rotated at 900 rpm overnight at 65℃. The DNA fragments were purified with Zymo genomic DNA clean&concentrator kit and eluted with 20 μL nuclease-free water. Proceed with tagmentation for 50ng ChIP-DNA in each 200 μL PCR tube using 5 μL Tn5 transposase at 55℃ for 10 min to obtain a 0.1-1kb tagmentated DNA profile. Purified the tagmentated DNA with DNA cleanup columns and immobilized with the pre-blocked and pre-cleaned M280 streptavidin dynabeads in final 1X binding and washing buffer at RT for 45 min with 15 rpm rotation. The supernatant was discarded and the beads were washed 5 times with 500 μL pre-warmed at 37℃ 0.5% SDS/2X SSC buffer and the beads were resined in 30 μL elution buffer. A 11-13 cycles on-beads PCR was performed to construct the in situ ChIA-PET libraries and the PCR products were purified with 1X AMPure beads. These final libraries were then subjected to a 0.6X-0.8X double-size selection using AMPure beads and paired-end sequencing (2 x 150bp) using Illumina NovaSeq 6000.