Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Pregnant females were euthanized using CO2 and the embryos removed from the womb and stored on a 10cm dish filled with chilled 1xPBS. Testicles were removed from the embryos, placed in an individual 15ml falcon tube with 3mls of 0.25 % Trypsin with 3μl of DNase I 1Unit/1μL (Life Technologies). Testes were incubated for 15mins at 37C. After incubation the cells were agitated into suspension gently by pipetting. The trypsin was then quenched using 5mL DMEM/10% FBS (Life Technologies). The cells were centrifuged at 278g for 5 minutes and resuspended in 500uL FACS buffer (1xPBS 1% BSA). 7-AAD was added at a 1:50 dilution (BD Biosciences) and the cells strained through BD FACS tubes (Corning) before analysis. GFP positive cells were sorted for ChIP. The ChIP-seq protocol was adapted from published sources (Ng et al., 2013, Pastor et al., 2014). FACS sorted cells from four male, E13.5 embryos were diluted to 292μL with room temperature 1xPBS. 8.11μL 37% Formaldehyde (Sigma) was added and the sample was incubated 10 minutes at room temperature with rocking. 48.8μL of 1M glycine was then added to yield a final concentration of 0.14M and the samples were quenched 30 minutes with rocking. Cells were then spun 425g for 10 minutes at RT. The cell pellet was flash frozen. After thawing, the cells were resuspended in 300μL Lysis buffer (50mM Tris-Cl pH 8.0, 20mM EDTA pH 8.0, 0.1% SDS, 1x Complete Protease Inhibitor (Roche)) and incubated on ice ten minutes. Samples were then sonicated by Covaris S2 (Intensity 5, cycles/burst=200, duty cycle=5%, 10x30s on 30s off sonication). Samples were spun 14000g 10 minutes to remove insoluble material. The soluble sample was diluted to 600μL with dilution buffer (16.7mM Tris pH 8, 0.01% SDS, 1.1% TritonX-100, 1.2mM EDTA, 167mM NaCl) and 10% of material was saved as input. Sample was precleared with 30μL Protein A Dynabeads (Life Technologies) and preincubated 1hr. The cleared material was incubated with 1μL L anti-H3K36me3 antibody (Abcam Ab9050) overnight. The samples were incubated with 30μL Protein A Dynabeads and the precipitated material was recovered with a magnet. The beads were washed 2x4 minutes with Buffer A (50mM HEPES pH 7.9, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 140mM NaCl), 2x4 minutes with Buffer B (50mM HEPES pH 7.9, 0.1% SDS, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 500mM NaCl) and 2x4 minutes with 10mM Tris/1mM EDTA. Bound material was eluted with 100μL Elution buffer (50mM Tris pH 8.0, 1mM EDTA, 1% SDS) at 65 C for ten minutes and then eluted a second time with 150μL elution buffer. The input samples were thawed and diluted with 200μL buffer. Crosslinking of ChIP and input samples was reversed by incubating 16 hours at 65C. Samples were cooled and treated with 1.5μl of 10mg/mL RNaseA (PureLink RNAse A, Invitrogen #12091-021) for thirty minutes at 37C. 100μg of Proteinase K was then added and the samples treated for 2hr at 56C. The samples were then purified using a Qiagen MinElute kit. Samples were amplified by a SeqPlex DNA Amplification kit (Sigma) and then converted to libraries using an Ovation Rapid Library kit.