Illumina HiSeq 1000 paired end sequencing; Germ cell epigenetic instability in the Mtrr mouse model of transgenerational epigenetic inheritance
Germ cell epigenetic instability in the Mtrr mouse model of transgenerational epigenetic inheritance
Sperm from the cauda epididymis and vas deferens were collected from 16-20 week-old fertile male mice using the technique previously described (Hisano et al., 2013) with the following amendments. Sperm were released for 20 minutes at 37◦C in Donners Medium (25 mM NaHCO3, 20 mg/ml BSA, 1 mM sodium pyruvate and 0.53% (vol/vol) sodium dl-lactate in Donners stock: 135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2 and 30 mM HEPES). Samples were centrifuged at 500xg (21◦C) for 10 minutes. The supernatant was then transferred and centrifuged at 3000rpm (4◦C) for 15 minutes. The majority of supernatant was discarded and samples centrifuged at 3000rpm (4◦C) for 5 minutes. Further supernatant was discarded and sperm centrifuged at 12000xg for 1 minute. Sperm were stored at -80◦C. Solution A (75 mM NaCl pH 8; 25 mM EDTA) and Solution B (10 mM Tris- HCl pH 8; 10 mM EDTA; 1% SDS; 80 mM DTT) were added to the sperm samples followed by RNAse A incubation (37◦C, 60 minutes) and Proteinase K incubation (overnight, 55◦C) (Radford et al., 2014). DNA was extracted using phenol/chloroform/isoamyl alcohol mix (25:24:1) (Sigma-Aldrich) as per the manufacturers instructions. DNA was precipitated using 10M ammonium acetate, glycogen (0.1 mg/ml), and 100 % ethanol, followed by incubation at -80◦C for at least 30 minutes. DNA was collected by centrifugation (13000 rpm, 30 minutes). The pellet was washed twice in 70 % ethanol and air-dried prior to resuspension in TE buffer. DNA quality and quantity was confirmed using gel electrophoresis and QuantiFluor dsDNA Sample kit (Promega) as per the manufacturer's instructions. MeDIP-seq libraries were generated as previously described (Radford et al.,2014; Senner et al., 2012). A total of 3μg of sperm DNA was sonicated using a Diagenode Bioruptor UCD-200 to 200-700bp fragments. DNA was then end-repaired, dA-tailed and pre-annealed paired-end adapters ligated using the NEBNext DNA Library Prep Master Mix for Illumina kit (NEB). After each step the DNA was cleaned using Agencourt AMPure XP SPRI beads (Beckman Coulter). MeDIP was performed in triplicate for each sample, with 500ng of DNA per IP. An input sample was reserved for each sample. MeDIP was performed using 1.25μl of anti-5mC antibody (Eurogentec BI-MECY-0100), the specificity of which has been previously confirmed (Radford et al., 2014). The immuno-precipitated fraction was isolated using Dynabeads M-280 sheep anti-mouse IgG bead (Invitrogen 112-01D). The unbound fraction was retained. The triplicate IPs for each sample were then pooled and purified using MinElute PCR Purification columns (Qiagen). Libraries were amplified by PCR using Phusion High-Fidelity PCR Master Mix (12 cycles) and barcoded using iPCR tag primers. Libraries were purified using Agencourt AMPure XP SPRI beads. The efficiency of the IP was verified using qPCR comparing pre-amplification input and IP fractions for regions of known methylation status (methylated: Nanog, H19 ICR, unmethylated: H1t, TsH2B ) (Lambrot et al., 2013). Library concentrations were estimated using the Kapa Library Quantification kit (Kapa Biosystems). Fragment size and library concentration were further verified by running on an Agilent High Sensitivity DNA chip on an Agilent 2100 BioAnalyzer.