Ab171870

ChIP was performed using 10 E9.5 embryos or 1 × 10⁶ ES cells per experiment. After recovery, embryos were treated with collagenase type I (Stemcell Technologies, 07902) at 0.125% for 1 hour at 37°. Then, embryos were desegregated using a pipette and washed with cold phosphate-buffered saline. Samples were fixed, and protein-DNA complexes were cross-linked by treatment with 1% formaldehyde (Pierce, 289069) for 15 min rocking at room temperature. To stop fixation, glycine (Nzytech, MBO1401) was added to a final concentration of 125 mM during 10 min. Next, ChIP was performed using the ChIP-IT High Sensitivity kit (Active Motif, 53040), following the manufacturer’s instructions. DNA was sheared into fragments ranging from 200 to 1000 bp using a sonicator (Diagenode Bioruptor Water Bath Sonicator, 30 s on 30 s off for 30 min). Immunoprecipitations were carried out using rabbit polyclonal anti-OCT4 antibody (Abcam, ab19857), and anti-Rabbit immunoglobulin G polyclonal antibody (Abcam, ab171870) was used as negative control. Enrichment was measured by qPCR. A fragment from the Nanog promoter was used as a positive control (27 (link), 29 (link)), and genomic fragments from the loci of Anks1b, Smg6, and Tiam1 were used as negative controls (59 (link)) after checking they did not contain OCT4 bound peaks (27 (link)). qPCR primers used are listed in data file S4.

ChIP experimental protocol was performed as previously described.⁴⁸ (link)^,51 (link) In brief, cell lysate was sonicated and then immunoprecipitated overnight using MLL3, H3K4me1 (2 μg for 25 μg of chromatin, ab8895) or non-specific IgG (1:1,000, ab171870, Abcam) antibodies as control. The next day samples were incubated with protein beads to form the protein-antibody-bead complex. After stringent washing, immunoprecipitated DNA was isolated used for qPCR. Anti-MLL3 was generated against the aa 443-590 (α-MLL3-NTD) or aa 2951-3091 (α-MLL3-MR) protein. The primers of the enhancer region of DACT2 were shown as follows: forward: 5′-ACAGGACTGGCTAAACGCAA-3′ and reverse: 5′-AGGTGCAAATGCTGCCCTAT-3′.¹⁸ (link)

To verify the binding of circ-0000512 to miR-622, the Magna RIP kit (Millipore, Bedford, Massachusetts, USA) was used in line with specific instructions in RIP assay. In short, MDA MB 231 and Hs578T cells (1×10⁷ cells) were collected and lysed with the RNA lysis buffer (100 µL). Later, 200 µL cell lysate was harvested and incubated for 12 hours at 4°C with the RIP immunoprecipitation buffer that contained protein A/G sepharose beads conjugated with IgG antibody (ab171870, Abcam, Shanghai, China) or Ago-2 antibody (ab186733, Abcam, Shanghai, China). Afterwards, the RNeasy Mini Kit (Qiagen, Valencia, California, USA) was employed to extract the immunoprecipitated RNA. Subsequently, cDNA was prepared from RNA through reverse transcription using the reverse transcription kit (Applied Bio-systems, Foster City, California, USA). The immunoprecipitated circ-0000512 and miR-622 levels were determined by qRT-PCR.
In the RIP assay to verify the binding of circ-0000512 to PD-L1 protein, 200 µL cell lysate was incubated for 12 hours at 4°C with RIP immunoprecipitation buffer that contained magnetic beads conjugated with anti-IgG or PD-L1 antibody. Later, the beads were treated with 0.5 mg/mL proteinase K (Beyotime, Shanghai, China) for 30 min at 55°C to remove proteins. As described above, the extraction, reverse transcription and quantification of circ-0000512 were performed sequentially.

After transfection, cells (3 × 10⁶ cells/sample) were cross-linked in 1% formaldehyde for 10 mins at room temperature, and then washed with PBS for two times and lysed with SDS lysis buffer (Upstate Biotechnology, Lake Placid, NY, USA). The lysates were then subjected to an ultrasonic bath for sonication (Bioruptor, Diagenode). After centrifugation at 15,000 × g for 10 mins, the supernatants were diluted with ChIP dilution buffer (Upstate Biotechnology) and were immunoprecipitated with rabbit anti-H2BK56ac (2 µg, ab195494, Abcam, Cambridge, UK) overnight at 4°C. The normal anti-IgG antibody (2 µg, ab171870, Abcam) was used as a control of immunoprecipitation. The beads were washed in low-salt for 5 mins at 4°C, and were washed twice in 1 × TE (Upstate Biotechnology) for 2 mins at room temperature. The DNA was eluted from the beads were treated as Liu et al described.19 (link) The serial dilutions of the 10% input DNA (1:4, 1:20, 1:100 and 1:500) were analyzed by using RT-qPCR.