GSM9058598: Ly3_IgG rep1; Homo sapiens; ChIP-Seq
Firstly, harvested cells were washed in PBS. For the first crosslinking step, each cell pellet was resuspended in 1 ml of PBS containing 4µl of disuccinimidyl glutarate (DSG, Thermo Fisher Scientific: 20593) and incubated for 45 minutes on a rotator. Samples were then washed twice with PBS before second cross-linking with 1080µl of 37% formaldehyde in 40 ml of PBS and left on the rotator at room temperature for 10 minutes. The reaction was stopped using 2.5 ml of 2M glycine. Samples were washed twice with PBS and resuspended in 2 ml of SDS lysis buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 10% SDS) with protease inhibitors and centrifuged at 1500 rpm for 5 minutes at 4°C. Subsequently, 2 ml of ChIP buffer (2 parts of SDS-lysis and 1 part of Triton-dilution buffer: 100 mM Tris-HCl pH 8.6, 100 mM NaCl, 5 mM EDTA, 5% Triton X-100) was added, and samples were split equally into two different 15 ml tubes for sonication. Cells were sonicated for 25 minutes at high output intensity at 4°C until they became clear and then centrifuged at 14,000 rpm for 30 minutes. The supernatant was pre-cleared with 60µl of protein A/G sepharose beads (Sigma Aldrich: 11719408001 and 11719406001) or 20ul of ChIP-grade magnetic beads (Thermo Scientific: 26162) at 4°C on a rotator for 2 hours. For input, 10% of the supernatant volume was set aside from the centrifuged samples. The remaining sample volume was divided equally into different tubes for incubation with 10 ug of primary antibody overnight on a rotator at 4°C. The antibodies for different proteins of interest and IgG control were added in equal amounts (Supp. Table 2). The next day, 60 μl of protein A/G sepharose beads or 20ul of ChIP-grade magnetic beads were added to the samples and incubated for 4 hours at 4°C. After discarding the supernatant upon centrifugation or a magnetic stand, the beads in the tubes were sequentially washed in 4 different buffers before the elution step. Purified DNA samples were sent to NovogeneAIT Genomics facility for ChIP-seq. Briefly, the DNA fragments were repaired, A-tailed and further ligated with Illumina adapter. The final DNA library was obtained by size selection and PCR amplification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries are pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required.