developmetal stage: young adult genotype: wild type strain: BN196
Extracted molecule
genomic DNA
Extraction protocol
DamID strains were synchronized from eggs prepared by hypochlorite treatment of gravid adults fed with OP50 E. coli and left to hatch overnight at 20ºC in S-medium. Then, from each strain, approximately 35000 L1 larvae were grown in 50mL S-medium containing GM119 E. coli. Cultures were grown with continuous agitation (180rpm) at 20ºC for 53 h. Worms were frozen at -80ºC until further processing. Methylated genomic DNA was purified and amplified from 30 mg worms. Briefly, 2.5μg of gDNA isolated with DNAeasy kit (Qiagen), was digested with 10 units DpnI (New England Biolabs) overnight in 10μL to cut at GAmTC sites. DpnI was inactivated (80ºC, 20 min) and then, gDNA was ligated to double-stranded adaptors with 5 units T4 DNA ligase (5U/μL, Roche) in a volume of 20μL. After inactivation of the ligase (65ºC, 10 min), DNA fragments were digested with 5 units DpnII (New England Biolabs) in a final volume of 80µl to cut unmethylated GATC sites thereby preventing PCR amplification of unmethylated gDNA. Methylated DNA was amplified using adaptor-specific primers. PCR reactions were performed using 1 µl PCR Advantage enzyme mix (50x) (Clontech), 10μL of DpnII digestion and the following program: (1) 68ºC 10 min, (2) 94ºC 1 min, (3) 65ºC 5 min, (4) 68ºC 15 min, (5) 94ºC 1 min, (6) 65ºC 1 min, (7) 68ºC 10 min, (8) Go to step 5 3x, (9) 94ºC 1 min, (10) 65ºC 1 min, (11) 68ºC 2 min, (12) go to step 9 20x. To achieve sufficient material, 2 PCR reactions from the same DpnII digestion were pooled and purified with Qiaquick PCR purification kit (Qiagen).
Label
Cy3
Label protocol
Amplified Dam methylated DNA was labeled by the Roche Nimblegen Service Laboratory.
developmental stage: young adult genotype: wild type strain: BN218
Extracted molecule
genomic DNA
Extraction protocol
DamID strains were synchronized from eggs prepared by hypochlorite treatment of gravid adults fed with OP50 E. coli and left to hatch overnight at 20ºC in S-medium. Then, from each strain, approximately 35000 L1 larvae were grown in 50mL S-medium containing GM119 E. coli. Cultures were grown with continuous agitation (180rpm) at 20ºC for 53 h. Worms were frozen at -80ºC until further processing. Methylated genomic DNA was purified and amplified from 30 mg worms. Briefly, 2.5μg of gDNA isolated with DNAeasy kit (Qiagen), was digested with 10 units DpnI (New England Biolabs) overnight in 10μL to cut at GAmTC sites. DpnI was inactivated (80ºC, 20 min) and then, gDNA was ligated to double-stranded adaptors with 5 units T4 DNA ligase (5U/μL, Roche) in a volume of 20μL. After inactivation of the ligase (65ºC, 10 min), DNA fragments were digested with 5 units DpnII (New England Biolabs) in a final volume of 80µl to cut unmethylated GATC sites thereby preventing PCR amplification of unmethylated gDNA. Methylated DNA was amplified using adaptor-specific primers. PCR reactions were performed using 1 µl PCR Advantage enzyme mix (50x) (Clontech), 10μL of DpnII digestion and the following program: (1) 68ºC 10 min, (2) 94ºC 1 min, (3) 65ºC 5 min, (4) 68ºC 15 min, (5) 94ºC 1 min, (6) 65ºC 1 min, (7) 68ºC 10 min, (8) Go to step 5 3x, (9) 94ºC 1 min, (10) 65ºC 1 min, (11) 68ºC 2 min, (12) go to step 9 20x. To achieve sufficient material, 2 PCR reactions from the same DpnII digestion were pooled and purified with Qiaquick PCR purification kit (Qiagen).
Label
Cy5
Label protocol
Amplified Dam methylated DNA was labeled by the Roche Nimblegen Service Laboratory.
Hybridization protocol
Amplified Dam methylated DNA was hybridized by the Roche Nimblegen Service Laboratory.
Scan protocol
Amplified Dam methylated DNA was scanned by the Roche Nimblegen Service Laboratory.
Description
EMR3 Methylated DNA
Data processing
Raw data were analyzed using MA2C software that normalized the log2 ratio (log2 [Dam::fusion/GFP::Dam control]) of each probe based on the probe behavior estimated by its GC content, and then smoothed the value by assigning the median across sliding windows of 300 bp. The resultant values are MA2C scores.
