GSM9557094: Patient104,CD14+,AS1-input(input); Homo sapiens; OTHER
Cells were harvested and washed with cold PBS. Nuclei were isolated using ATAC lysis buffer according to standard ATAC-seq protocols. Approximately 50,000 nuclei were used for downstream transposition reactions. ATAC-seq libraries were prepared following the protocol described by Buenrostro et al. with minor modifications. Isolated nuclei were subjected to Tn5 transposition reaction. Following DNA fragmentation and purification, libraries were amplified by PCR for 14 cycles. Library fragment size distribution was assessed using an Agilent Bioanalyzer. Libraries were sequenced on an Illumina HiSeq 2500 platform. Cells were crosslinked with 1% formaldehyde and lysed. Chromatin was fragmented by sonication to generate DNA fragments of approximately 200–500 bp. Chromatin Isolation by RNA Purification (ChIRP) was performed according to the manufacturer's protocol (APE×BIO, China). Biotin-labeled probes targeting IRF1-AS1 were hybridized with chromatin lysates at 37°C. RNA–protein–DNA complexes were captured using streptavidin-coated magnetic beads. After stringent washing and reverse cross-linking, DNA was purified and used for library preparation. Libraries were sequenced on an Illumina platform. An irrelevant probe was used as a negative control. Crosslinked cells were lysed and chromatin was sheared by sonication at 4°C using a Bioruptor 300 to generate fragments of approximately 200–400 bp. Soluble chromatin was precleared with protein A agarose beads prior to immunoprecipitation. The precleared chromatin was incubated overnight at 4°C with antibodies. After washing, immunoprecipitated chromatin was eluted and reverse cross-linked at 65°C for 12 hours. DNA was purified by phenol–chloroform extraction followed by ethanol precipitation. Sequencing libraries were prepared using standard Illumina-compatible protocols and sequenced on an Illumina platform.