GSM2493805: Donor 2- ChIPseq of H3K4me1 in purified primary AT2 cells; Homo sapiens; ChIP-Seq
ChIP-seq on AT2 cells was performed as previously described(Marconett et al. 2013). ChIP-Seq of AT2 cells from Donor 1 and Donor 2 in this Study. ChIP-seq of human AT2 cells from Donor1 and Donor2 in this study: Human AT2 cells underwent ChIP-seq using a modified version of the Sigma ChIP-it kit (Sigma). Specifically, 2 million human AT2 were used per ChIPseq of H3K27Ac (Cat # 39135, ActiveMotif), H3K4me1 (Cat # 39297, ActiveMotif). 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. All ChIP-seq 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. Whole genome bisulfite sequencing (WGBS) of AT2 cells from Donor 1 and Donor 2 in this Study. Total DNA from 8x10^6 Donor 1 and Donor 2 AT2 cells in this study was isolated using the Illustra Triple Prep kit (GE LifeSciences, Piscataway NJ). Four μg of sample genomic DNA was sonicated using a Covaris S2 to an average molecular weight of 150 bp.   Achievement of the desired size range was verified by Bioanalyzer (Agilent) analysis. Fragmented DNA was repaired to generate blunt ends using the END-It kit (Epicentre Biotechnologies, Madison, WI) according to manufacturer’s instructions.  Following incubation, the treated DNA was purified using AmpureX beads from Agencourt.  In general, magnetic beads were employed for all nucleic acid purifications in the following protocol.  Following end repair, A-tailing was performed using the NEB dA-tailing module according to manufacturer’s instructions (New England Biolabs, Ipswich, MA). Adapters with a 3’ ‘T’ overhang were then ligated to the end-modified DNA.  For whole genome bisulfite sequencing, modified Illumina paired-end (PE) adapters were used in which cytosine bases in the adapter are replaced with 5-methylcytosine bases. Ligation was carried out using ultrapure, rapid T4 ligase (Enzymatics, Beverly, MA) according to manufacturer’s instructions.  The final product was then purified with magnetic beads to yield an adapter-ligation mix. Prior to bisulfite conversion, bacteriophage lambda DNA that had been through the same library preparation protocol described above to generate adapter-ligation mixes was combined with the genomic sample adapter ligation mix at 0.5% w/w.  Adapter-ligation mixes were then bisulfite converted using the Zymo DNA Methylation Gold kit (Zymo Research, Orange, CA) according to the manufacturer’s recommendations. Final modified product was purified by magnetic beads and eluted in a final volume of 20 ul. Amplification of one-half the adapter-ligated library was performed using Kapa HiFi-U Ready Mix for the following protocol: 98° 2’, Then six cycles of: 98° 30”, 65° 15”, 72° 60”, with a final 72° 10’ extension, in a 50 ul total volume reaction. Final library product was examined on the Agilent Bioanalyzer, then quantified using the Kapa Biosystems Library Quantification kit according to manufacturer’s instructions.   Optimal concentrations to get the right cluster density were determined empirically but tended to be higher than for non-bisulfite libraries.  Libraries were plated using the Illumina cBot and run on the Hi-Seq 2000 according to manufacturer’s instructions using HSCS v 1.5.15.1. Donor 1 samples underwent Paired End 100 cycling; Donor 2 and 3 samples underwent Paired End 75 cycling. Image analysis and basecalling were carried out using RTA 1.13.48.0, deconvolution and fastq file generation were carried out using CASAVA_v1.7.1a5. Alignment to the genome was carried out using bsmap V 2.5. Libraries were prepared at the USC Epigenome Core as previously  described (Marconett, 2013).