Name: GSM7181652
Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Chip-seq was carried out as described in Deluca et al (2020). Since we worked with cells with various ploidy levels spanning from 2C to 512C, we standardized input to the number of genomes rather than the number of cells. Specifically, for each IP, the equivalent of 500,000 2C sorted nuclei (i.e. 1 million genomes) were mixed with a specific amount of fixed mouse 3T3 cells as a spike-in (the same amount across samples). Mixed samples were then sonicated to acquire mono-nucleosome fragments: nuclei were resuspended in 100 µl of Nuclei Resuspension Buffer (all recipes at end), and were sonicated in a Bioruptor Pico instrument with 22 cycles of 30 s on, 30 s off. Fragment sizes were confirmed with the Bioanalyzer (Agilent). Subsequently, 900 µl of Dilution Buffer was added to the 100 ul of mononucleosome fragments, then 1% of each sample was set aside to be used for input controls. To prepare antibody-conjugated beads, 10 µl of antibody (either H3K27ac, H3K27me3, or H3K9me3) was preincubated with 25 µl of a 1:1 mix of proteinA:proteinG dynabeads, then washed twice with PBS + 0.02% Tween 20 (PBST). To bind mononucleosome fragments that harbored the target antigen, we combined antibody-conjugated beads with the chromatin extract(s) on a rocker at 4°C overnight. The next day, the beads were washed twice for 15 min each with Wash buffer A, Wash buffer B, Wash buffer C, and TE buffer. Chromatin was eluted and reverse-crosslinked by incubating at 65°C overnight with Direct Elution Buffer (DEB). For input samples (set aside earlier), cross links were reversed by adding NaCl to 300 mM, then adding DEB to equalize the volume between inputs and IPs, then incubating at 65°C overnight. All samples were then incubated for 30 min at 37°C with 0.3 mg/ml RNAse A, and subsequently for 2 hr at 55°C with 0.6 mg/ml proteinase K. DNA was then extracted with phenol:chloroform, precipitated with NaAc/ethanol, washed with 70% ethanol wash, and resuspended samples in 10 µl water and of which all 10 µl was used for library preps. Recipes: Nuclei Resuspension Buffer (50 mM TrisHCl pH 8.0, 10 mM EDTA, 1% SDS, and proteinase inhibitor cocktail (Roche)). Dilution Buffer (15 mM TrisHCl pH 8.0, 1 mM EDTA, 1% Triton X-100, 150 mM NaCl). Wash buffer A (20 mM TrisHCl pH8.0, 2 mM EDTA, 0.1% SDS, 1% Triton X100, 150 mM NaCl), Wash buffer B (20 mM TrisHCl pH8.0, 2 mM EDTA, 0.1% SDS, 1% Triton X100, 500 mM NaCl), Wash buffer C (10 mM TrisHCl pH8.0, 1 mM EDTA, 1% NP40, 1% Sodium deoxycholate, 0.25M LiCl), TE buffer (10 mM TrisHCl pH8.0, 1 mM EDTA). Direct Elution Buffer (DEB) (10 mM TrisHCl pH 8.0, 300 mM NaCl, 5 mM EDTA, 0.5% SDS). The Illumina sequencing library was prepared with Takara Bio / Rubicon ThruPLEX DNA-seq kit and sequenced on an Illumina NextSeq 500 to yield 75 bp, single-end reads.