title: GSM2463914: Donor1_H3K4me1_D6; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIPseq of human AEC cells: Human AEC cells underwent ChIPseq using a modified version of the Sigma ChIP-it kit (Sigma). Specifically, 2 million human AEC were used per ChIPseq of H3K27Ac (Cat # 39135, ActiveMotif), H3K4me1 (Cat # 39297, ActiveMotif), and H3K9Ac (Cat # 39137, ActiveMotif). 10 million cells were used for ChIPseq of CTCF (Cat # 2899S, Cell Signaling Technology). All cells were crosslinked with 3.75% formaldehyde at 25C for 5 minutes with gentle nutation. Fixation was quenched with 2.5mM glycine, then cells frozen at -80c until differentiation of all time points was complete. Each sample underwent nuclei fractionation using cell lysis buffer prepared per manufacturer’s recommendations. Upon nuclei isolation, cells were sonicated into fragments (average size 300bp) using a Bioruptor2000 (Diagenode) with 30-second pulses for a total of 30 minutes of sonication (15 on, 15 off). StaphSeq (Cat # S6576, Sigma) was used for antibody binding. All ChIPs were verified for enrichment by site-specific PCR prior to sequencing using the active region of PGDH for enhancer marks, and for CTCF the canonical binding site in ZNF333. All ChIPseq samples underwent library preparation and sequencing at the USC Epigenomic Core. Samples were multiplexed using adapter barcoding and underwent single-end 50bp sequencing using the IlluminaHiSeq2000.   FAIREseq: FAIRE input material was isolated from the combined supernatant (unbound fraction) of all histone modification ChIPseq samples as described previously (Giresi et al., 2007 Genome Research). Samples underwent reverse-crosslinking and proteinase K digestion at 65C overnight, followed by phenol/chloroform extraction. Sequencing was performed at the USC Epigenomic Core. Samples were multiplexed using adapter barcoding and underwent single-end 50bp sequencing using the IlluminaHiSeq2000.   ATACseq: Freshly isolated cells from Donor 4 were subjected to transposon insertion as described in detail previously (Buenrostro et al., 2015 Curr. Protoc Mol Biol., Buenrostro et al., 2013 Nature Methods). 32,000 basal cells and 50,000 macrophage, fibroblast, and AT2 cells underwent transposase insertion. In brief, cells were pelleted at 4°C, washed in cold PBS, and lysed in 50 uL of cold lysis buffer (10mM Tris-HCl pH=7.4, 10mM NaCl, 3mM MgCl2, 0.25% IGEPAL). Intact nuclei were pelleted, resuspended in 50 uL of transposition reaction mix (1X TD Buffer, 5% v/v Tn5 transposase, Illumina) and immediately incubated at 37°C for 30 minutes. Transposed DNA was extracted in a final volume of 10 uL using the Qiagen MinElute Kit. To generate ATAC-seq libraries, transposed DNAs were first amplified by 5 cycles of PCR (NEBNExt High-Fidelity PCR Master Mix, 750nM forward and reverse customized Nextera PCR primers, 50 uL final volume). PCR amplification with adapter sequences was performed as previously described (Quail et al., 2014, BMC Genomics). To reduce PCR bias, the total number of PCR amplification cycles was determined by qPCR. Briefly, 5 uL of the PCR reaction was amplified by 20 cycles of qPCR (NEBNExt High-Fidelity PCR Master Mix, 420nM customized forward and reverse Nextera PCR primers, 0.6X SYBR Green I, 15 uL final volume). The total number of PCR cycles was determined by using the qPCR amplification plot and taking the cycle number corresponding to one-third the maximum fluorescence intensity. The remaining volume of the PCR reaction was further amplified according to the total number of cycles determined by qPCR. ATAC-seq libraries were purified by performing a 1.5X bead clean with Agencourt AMPure XP Magnetic Beads and analyzed for quality control using a BioAnalyzer High-Sensitivity DNA Analysis kit.  Samples underwent single-end 50-bp sequencing on the Illumina NextSeq at the USC Epigenome center. To generate ATAC-seq libraries, transposed DNAs were first amplified by 5 cycles of PCR (NEBNExt High-Fidelity PCR Master Mix, 750nM forward and reverse customized Nextera PCR primers, 50 uL final volume). To reduce PCR bias, the total number of PCR amplification cycles was determined by qPCR. Briefly, 5 uL of the PCR reaction was amplified by 20 cycles of qPCR (NEBNExt High-Fidelity PCR Master Mix, 420nM customized forward and reverse Nextera PCR primers, 0.6X SYBR Green I, 15 uL final volume). The total number of PCR cycles was determined by using the qPCR amplification plot and taking the cycle number corresponding to one-third the maximum fluorescence intensity. The remaining volume of the PCR reaction was further amplified according to the total number of cycles determined by qPCR. ATAC-seq libraries were purified by performing a 1.5X bead clean with Agencourt AMPure XP Magnetic Beads and analyzed for quality control using a BioAnalyzer High-Sensitivity DNA Analysis kit.
attributes.tag: GEO Accession
attributes.value: GSM2463914