GSM6913972: GFP-Dam_intestine_rep2; Caenorhabditis elegans; OTHER
Genomic DNA (gDNA) extraction from ~30 μl L4s was performed by 5 rounds of rapid freeze/thaw cycles in liquid nitrogen and 37°C water bath to break the worm cuticle followed by purification using a DNeasy Blood and Tissue Kit (QIAGEN #69504). gDNA was eluted in two rounds in a total volume of 300 μl and precipitated using 0.1x volume NaAc 3M and 2.5x volume 96% ethanol. The final concentration after resuspension in 10 μl of 10mM Tris-Cl was measured by QUBIT. To prepare the libraries, 200 ng gDNA were digested using 10 units DpnI (NEB #R0176S; cuts methylated GATC) in 10 μl for 6 hours at 37°C in a thermocycler. Next, DpnI was inactivated by heating to 80°C for 20 min. Double-stranded adaptors were prepared by heating a mixture of 50 μl primer AdRt (5'-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA, 100 μM) and 50 μl AdRb (5'-TCCTCGGCCGCG; 100 μM) to 95°C and cooling slowly to room temperature. Next, 0.8 μl of the double-stranded adapters were ligated to the DpnI-digested gDNA in a volume of 20 μl at 16°C in a thermocycler overnight using T4 DNA ligase (Roche #10799009001), which later was inactivated by incubation for 10 min at 65°C. The ligated gDNA was purified with 1.8x volume Agencourt AMPure XP® beads (Beckman Coulter; #A63880) using a magnetic particle concentrator, washed with 180 μl of freshly prepared 70% ethanol and eluted in 20 μl elution buffer (EB; 10 mM Tris-Cl, pH 8.5). Unmethylated GATC sites were digested with 10 units of DpnII (NEB #R0543S) in a volume of 50 μl for 1 h at 37°C. Next, DpnII was inactivated by heating to 80°C for 20 min. The digested gDNA was re-purified with AMPure XP® beads as above and eluted in 25 μl. Methylated gDNA fragments were amplified using Taq DNA polymerase with ThermoPol buffer (NEB #M0267) in 50 μl reactions containing 5 μl 10x PCR reaction buffer, 25 μl DpnII digested DNA, 1.25 μl primer AdR (5'-NNNNGTGGTCGCGGCCGAGGATC; 50 μM), 4 μl dNTP mix (each 2.5 mM), 1 μl polymerase. PCR parameters were 1) 68°C 10 min; 2) 94°C 1 min; 3) 65°C 5 min; 4) 68°C 15 min; 5) 94°C 1 min; 6) 65°C 1 min; 7) 68°C 10 min; 8) Go to step 5 and repeat three times; 9) 94°C 1 min; 10) 65°C 1 min; 11) 68°C 2 min; 12) Go to step 9 and repeat 19 times. Five μl of each PCR reaction were analyzed by agarose gel electrophoresis: successful amplification of methylated DNA was confirmed by the presence of a 400–1000 bp smear which was not observed in controls without DpnI or T4 DNA ligase. Finally, the PCR products were purified using QIAquick PCR Purification Kit (QIAGEN #28104), eluted in 30 μl EB and their concentration was measured using QUBIT. 400 ng of PCR fragments were used as input in the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370). The manufacturer's instructions were used with the following conditions. Size selection was performed by first adding 30 μl of resuspended SpeedBead Magnetic Carboxylate Modified Particles, Azide 0.05% (GE65152105050250) to the 100 μl ligation reaction. The beads were discarded and the supernatant was mixed with 60 μl resuspended SpeedBeads. Then, the supernatant was discarded and the beads were washed with ethanol and DNA was eluted in 17 μl of 10mM Tris. Eight PCR cycles were performed to amplify the samples. The quality of the samples was verified by agarose gel electrophoresis and QUBIT analysis as above.