GSM8395882: MSK-150, CD45+, non-mGAM, FOSL2, ChIPseq; Homo sapiens; ChIP-Seq
Freshly harvested cells were sorted, crosslinked in PBS with 1% formaldehyde for 10 min at room temperature, quenched with 0.125M glycine for 5 min and washed in PBS with protease inhibitors (Millipore Sigma, USA) three times before being snap frozen in cryotubes and sent to MSKCC's Epigenetics Research Innovation Lab for processing. Low input ChIP-seq was performed using the Low-Cell ChIP-Seq kit (Active Motif, USA) following the manufacturer's instructions, with the subsequent modifications. After thawing, the fixed cells were resuspended in 130uL of ChIP buffer, transferred into a microTUBE-130 (Covaris, USA) and sonicated using a Covaris E220 ultra-sonicator, with the following parameters: PIP=140, Duty Factor=5, CBP/Burst per sec=200, Time = 900s. After sonication, samples were transferred to siliconized micro-centrifuge tubes and centrifuged at 18,000g for 2 minutes at 4°C. After centrifugation, the supernatants were transferred to new labeled siliconized micro-centrifuge tubes. One percent of each sample was used as input. All samples and inputs were snap frozen and stored at -80°C. After the chromatin being processed following the manufacturer's recommendations, all samples and inputs were de-crosslinked overnight at 65°C. DNA was finally isolated using the ChIP DNA Clean & Concentrator kit (Zymo Research, USA) following the manufacturer's instructions. After quantification of the recovered DNA fragments, libraries were prepared using the ThruPLEX®DNA-Seq kit (Takara Bio Inc., Japan) following the manufacturer's instructions, purified with SPRIselect magnetic beads (Beckman Coulter), quantified using a Qubit Flex fluorometer (ThermoFisher Scientific, USA) and profiled using a TapeStation 2200 (Agilent Technologies, USA).