cell type: Sperm strain: AB developmental stage: Sperm [from adult males] medip antibody: Anti-5-methylcytosine antibody (10 ng/µl) medip antibody catalog#: Mab-006-100 medip antibody vendor: Diagenode sample preparation protocol: Zebrafish males were starved for 24 hrs and anesthetized in fish water with 0.016% tricaine (Sigma, Cat. No A-5040) immediately before sperm collection. Sperm was obtained with a capillary tube under gentle suction and flash frozen in liq. N2. Prior to DNA isolation, sperm was washed three times in T10N150E10 buffer (10 mM Tris-HCl pH 7.8, 150 mM NaCl,10 mM EDTA pH 8.0)
Growth protocol
Fish and embryos grown as per AAALAC certification, sperm collected as described in Draper, B.W., Moens, C.B. A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization. J. Vis. Exp. (29), e1395, DOI : 10.3791/1395 (2009).
Extracted molecule
genomic DNA
Extraction protocol
Embryos and sperm were lysed in lysis buffer, proteinase K treated and sonicated with a Bioruptor (Diagenode). DNA was purified by PCI and ethanol precipitation, and treated with RNase. DNA was re-sonicated to 300-1000 bp fragments. MeDIPs were performed using 4 µg DNA and 5 µl (10 ng/µl) 5-methylcytosine antibody. Samples were washed, deproteinized and DNA purified. Input DNA was fragmented and treated as above without any immunoprecipitation. MeDIP and input DNA was amplified (Sigma-Aldrich, WGA2), cleaned up, eluted and processed for array hybridization (Roche-Nimblegen).
Label
Cy5
Label protocol
Labeling done by Nimblegen as per normal service protocol
cell type: Sperm strain: AB developmental stage: Sperm [from adult males] antibody: none, input sample preparation protocol: Zebrafish males were starved for 24 hrs and anesthetized in fish water with 0.016% tricaine (Sigma, Cat. No A-5040) immediately before sperm collection. Sperm was obtained with a capillary tube under gentle suction and flash frozen in liq. N2. Prior to DNA isolation, sperm was washed three times in T10N150E10 buffer (10 mM Tris-HCl pH 7.8, 150 mM NaCl,10 mM EDTA pH 8.0)
Growth protocol
Fish and embryos grown as per AAALAC certification, sperm collected as described in Draper, B.W., Moens, C.B. A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization. J. Vis. Exp. (29), e1395, DOI : 10.3791/1395 (2009).
Extracted molecule
genomic DNA
Extraction protocol
Embryos and sperm were lysed in lysis buffer, proteinase K treated and sonicated with a Bioruptor (Diagenode). DNA was purified by PCI and ethanol precipitation, and treated with RNase. DNA was re-sonicated to 300-1000 bp fragments. MeDIPs were performed using 4 µg DNA and 5 µl (10 ng/µl) 5-methylcytosine antibody. Samples were washed, deproteinized and DNA purified. Input DNA was fragmented and treated as above without any immunoprecipitation. MeDIP and input DNA was amplified (Sigma-Aldrich, WGA2), cleaned up, eluted and processed for array hybridization (Roche-Nimblegen).
Label
Cy3
Label protocol
Labeling done by Nimblegen as per normal service protocol
Hybridization protocol
Hybridization done by Nimblegen as per normal service protocol
Scan protocol
Scanning done by Nimblegen as per normal service protocol
Data processing
log2 (MeDIP/Input) with biweight mean of values subtracted
