C. elegans strains and growth conditions
Animals were reared on E. coli grown on NGM (nematode growth medium) nutrient plates [39]. Bacterial strains used in this work as food for C. elegans are noted for each experiment.
OP50[39]: A uracil-auxotrophic E. coli strain with wild-type dam and dcm methylation systems. This strain has been a standard laboratory food source for C. elegans.
SCS110: An E. coli strain defective in both dcm and dam methylation: rpsL (Strr) thr leu endA thi-1 lacY galK galT ara tonA tsx dam dcm supE44D (lac-proAB) [F' traD36 proAB lacIqZDM15]. This strain provides a suitable food source for C. elegans while avoiding the presence of bacterial sequences with adenine methylation in eventual sequencing libraries.
SCS110(AmpR): SCS110 made ampicillin resistant by transformation with pUC18. This strain adds the ability to "switch" bacterial food sources in a culture by cultivation of previously OP50-fed populations with SCS110(AmpR)+ and ampicillin.
All worm strains were reared at 23°C unless otherwise stated. C. elegans strains used in the experiments were as follows:
N2: wildtype strain of C. elegans (Bristol isolate)[39]
PD5122 [ pha-1 ( e2123ts ) III; ccEx5122 ]: transgenic line expressing E. coli dam-GFP translational fusion and genomic C. elegans pha-1(+) gene from the extra-chromosomal array ccEX5122. Line PD5122 was established by microinjection of a mixture of plasmids pPD177.01 (Lig6682) and pC1 into pha-1(e2123ts) animals. pPD177.01 contains the myo-3 (body wall muscle) promoter driving E. coli dam fused to GFP. Two introns with C. elegans consensus sequences have been inserted into the dam gene to optimize expression in nematodes [28, 40]. A detailed description of pPD177.01 structure is shown in Additional File 1, Figure S1. pC1 carries the wildtype pha-1 coding region; non-transformed pha-1(e2123ts) animals are inviable at 23°C, while transformed animals carrying pC1 are viable, providing a strong selection [29].
PD3994 [ pha-1 ( e2123ts ) III; ccEx3994 ]: transgenic line expressing E. coli dam and genomic C. elegans pha-1(+) gene from the extra-chromosomal array ccEx3994. Line PD3994 was established by microinjection of plasmids pPD176.59 (Lig6649) and pC1(FD142), which contains the C. elegans genomic pha-1 gene [29] and is a selection marker for ccEx3994. pPD176.59 contains the E. coli dam gene driven by the myo-3 promoter and a single SV40 nuclear localization signal.
PD3995 [ pha-1 ( e2123ts ) III; ccEx3995 ]: transgenic line expressing E. coli dam and genomic C. elegans pha-1(+) gene from the extra-chromosomal array ccEx3995. Line PD3995 was generated by microinjection of a mixture of plasmids L7710, pRF4 (carrying the C. elegans rol-6[su1006] [41]), and pC1 into pha-1(e2123ts) animals. Plasmid L7710 contains the rol-6 promoter [42] driving the expression of a GFP-DAM translational fusion (rol-6::gfp-dam-unc-54 3' UTR). Attached to the 3' end of L7710 is the unc-54 3' UTR [28].
PD3997[ pha-1 ( e2123ts ) III; ccEx3997 ]: transgenic line expressing E. coli dam and genomic C. elegans pha-1(+) gene from the extra-chromosomal array ccEx3997. Line PD3997 was established by microinjection of a mixture of plasmids consisting of pC1, pRF4, and L7715 (vit-2::gfp-dam-unc-54 3' UTR).
Southern hybridization
Southern hybridizations were performed according to standard protocols. Briefly, RNase A-treated genomic DNA was subjected to one hour restriction digest by Dpn I, Mbo I, or Sau3A I, followed by phenol:chloroform extraction and ethanol precipitation. Restricted fragments were resolved on 1.4% agarose gels followed by transfer to Hybond-N+ membranes (Amersham Biosciences, Cat #RPN303B as recommended for capillary blotting under alkali conditions). An 808 bp radiolabeled probe containing the C. elegans 5S rDNA/SL1 (Spliced Leader sequence 1) was synthesized from a Bam H1 fragment of plasmid pPD98.38 using the RadPrime DNA Labeling System (Invitrogen, Cat #18428-11) with labeled α-32P dATP (MP Biomedicals, Cat #33002HD.5). Pre-hybridization and hybridization were in roller bottles using phosphate-SDS buffer (0.5 M phosphate buffer pH7.2, 1 mM EDTA pH8.0, 7% (w/v) SDS, 1% (w/v) BSA).
DNA extraction from synchronized animal populations
To generate synchronized worm populations, embryos were collected by treating gravid animals with a solution containing 1 M NaOH in 10% bleach for approximately 5-7 minutes or until adult cuticles were completely disintegrated [39]. Eggs were washed several times with M9 medium (22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl, 1 mM MgSO4) and distributed onto NGM plates containing a thin layer of SCS110 E. coli seeded the previous night. Synchronized populations were collected at the stage where the desired tissue mass would be greatest per animal (L1/L2 larvae for the myo-3 promoter in line PD3994; L4 larvae for the rol-6 promoter in line PD3995; and young/gravid adults for the vit-2 promoter in line PD3997). At no time were synchronized animals starved.
Synchronized animals were washed off NGM plates with chilled M9 medium, layered on a 5% sucrose solution, and pelleted by centrifugation at low speed. Pelleted animals were washed several times with chilled M9 and frozen as ~50 μL pellets at -80°C. It is important to note that throughout the harvesting procedure, animals were alive up to the time before freezing.
Genomic DNA was extracted using the following procedure. To each thawed ≈50 μl pellet was added 450 μl worm lysis buffer (0.1 M Tris pH 8.5, 0.1 M NaCl, 50 mM EDTA, 1% SDS), 1 μl 10 mg/ml glycogen, and 20 μL 20 mg/ml proteinase K in TE pH 7.4. The mixture was incubated at 62°C for 45 minutes, with intermittent vortexing. The mixture was extracted with 500 μl phenol, followed by 500 μl phenol:chloroform (1:1), and 500 μl chloroform. DNA was precipitated with 20 μl saturated ammonium acetate and 1 ml 100% ethanol, washed once with 500 μl ethanol, and resuspended in 50 μl TE pH 7.4. Each 50 μl sample was treated with 1 μl 10 mg/ml RNase A for one hour at 37°C. The reaction was terminated with 1× STOP buffer (1 M NH4Ac, 10 mM EDTA, 0.2% SDS) followed by phenol:chloroform/chloroform extraction and 100% ethanol precipitation. The final product was resuspended in 40-50 μl TE and used for DALEC library preparation.
in vitro methylation of N2 genomic DNA
N2 genomic DNA was methylated using the following 200 μl reaction mixture: 30.0 μl (≈20-30 μg) N2 genomic DNA, 0.5 μl 32 mM S-adenosyl methionine, 1.0 μl E. coli DAM (8 U/μl, NEB M0222S), 20.0 μl 10× DAM buffer, 148.5 μl dH2O. Following one hour incubation at 37°C, the reaction was terminated with 1× STOP buffer. To the terminated reaction mixture was added 1 μl 10 mg/ml glycogen followed by 500 μl phenol:chloroform extraction, 500 μl chloroform extraction, 100% ethanol precipitation, 0.5 ml 100% ethanol wash.
Dpn I digestion
Dpn I digestion was carried out in a 200 μl volume consisting of the following mix: 30 μl (≈20-30 μg) genomic DNA, 20 μl 10× buffer (NEB4), 10 μl Dpn I (20 U/μl, NEB #R0176), and 140 μl dH2O. The reaction mix was incubated for 1.5 hours at 37°C and terminated with 350 μl 1× STOP buffer. 1.0 μl 10 mg/ml glycogen was added to the mix followed by 500 μl phenol:chloroform extraction, 500 μl chloroform extraction, precipitation with 100% ethanol, wash with 0.5 ml 100% ethanol, and resuspension in 10 μl TE. (NOTE: Unless otherwise indicated, all enzymatic reactions described below used the same termination, extraction, and precipitation steps).
Ligation to Linker A
Linker A was purchased as two separate oligonucleotides (5'OH-CAAGCAGAAGACGGCATACGATCCTGAGTACACTATGTTCCGAC-OH3', 5'P-GTCGGAACATAGTGTAGCA-OH3') and hybridized by boiling in a flask of water for five minutes and allowing the water to cool to room temperature. Ligation to Linker A was carried out in a 50 μl reaction using the following mix: 10 μl Dpn I product, 1.5 μl of 0.05 mM Linker A, 11.0 μl dH2O, 25.0 μl 2× Quick Ligase Buffer, 2.5 μl Quick Ligase (NEB #M2200). The reaction was incubated for five minutes at room temperature followed by termination, extraction, precipitation, and resuspension of ligated products in 10 μl TE.
To increase the number of ligated molecules, we added a second ligation step using the following mix: 10 μl Quick Ligase product, 7 μl dH2O, 2 μl 10× ligase buffer, 1 μl T4 DNA ligase (2,000 U/μl; NEB #M0202). The reaction was allowed to proceed for 30 minutes at room temperature followed by termination, extraction, precipitation, and resuspension in 20 μl TE.
Mme I digestion
Linker A-ligated molecules were subjected to Mme I digestion using the following 200 μl reaction mix: 20 μl Linker A-ligated product, 20.0 μl 10× NEB 4, 0.3 μl 32 mM S-adenosyl methionine, 2.0 μl Mme I (2 U/μl; NEB R0637), 157.7 μl dH2O. The reaction was allowed to proceed for 1 hour at 37°C followed by termination, extraction, precipitation, and resuspension in 10 μl TE.
Ligation to Linker B
Linker B was purchased as two separate oligonucleotides (5'P-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTCGGTGGTCGCCGTATCATT-OH3', 5'OH-TCATCTTTCCCTACACGACGCTCTTCCGATCTNN-OH3') and hybridized using the same procedure as described for Linker A. Mme I products were ligated to Linker B using the following 50 μl reaction mix: 10.0 μl Mme I product, 1.0 μl of 0.05 mM Linker B, 5.0 μl 10× ligase buffer, 3.0 μl T4 DNA ligase (2,000 U/μl; NEB #M0202), 31.0 μl dH2O. Ligations were performed overnight using a PCR machine. Reactions were initiated at 8°C and stepped up to 16°C, with each degree increase in temperature held for two hours. Ligated products were extracted, precipitated, and resuspended in 10 μl TE.
Size selection
Linker A and Linker B ligated molecules were size fractionated on a 6% polyacrylamide:formamide denaturing gel (15% v/v 19:1 acrylamide:bis (40%), 1× TBE, 25% (v/v) 100% formamide, 42% w/v urea). Electrophoresis was performed in 0.5× TBE at 700 V for approximately 2.5-3 hours. The 116 nt single-stranded DNA product was cut out from the denaturing gel, using single-stranded oligonucleotides of sizes 95, 105, 114, 116, and 125 as size guides. Products were passively eluted from excised bands overnight in 0.3 M NaCl at 4°C, precipitated in 100% ethanol, and resuspended in 20 μl TE.
PCR amplification
PCR reactions were performed in a 50 μl reaction consisting of the following mix: 5 μl PAGE-purified template, 1.0 μl each of Solexa bridge amplification primers (1 μg/μl), 5 μl dNTP mix (2 mM each), 5 μl 10× NEB ThermoPol PCR buffer, 1 μl Taq polymerase (5 U/μl; NEB M0267), 32 μl dH2O. Reaction cycles were titrated to determine the linear range, typically 11-17 cycles of 45 s at 94°C, 30 s at 55°C, and 30 s at 72°C, with an initial denaturation step of 60 s at 94°C and a final extension step of 60 s at 72°C. PCR products were separated on 3% low melting point agarose gel (NuSieve Cat #50084). For a gel of approximately 10 inches long, electrophoresis at 103 V for approximately six hours gave superior resolution. The desired 116 bp dsDNA product was excised and recovered from the gel using the following steps. To each cut band was added 400 μl 1× STOP buffer, 1 μl glycogen and incubated in a 68°C water bath until the agarose was completely melted. To each tube of melted agarose was added 350 μl of 68°C phenol, quickly vortexed, spun 5-7 minutes, and the aqueous phase extracted (typically, a second 1-2 minute spin was required to completely remove residual agarose). Following extraction with 250 μl 1:1 phenol:chloroform and 250 μl chloroform, DNA was precipitated in 1 ml 100% ethanol, washed with 0.5 ml 100% ethanol, and resuspended in 15 μl TE for each 5 μl PCR template used.
Sequencing
Sanger sequencing was performed by Elim Biopharmaceuticals Inc. (Hayward, CA). High throughput sequencing of captured DAM tags was performed on the Solexa Genome Analyzer I.
in silico identification of DAM tags
We generated a database of all potential DAM tags with the structure 5'-(N)16GATC-3' from C. elegans genome version WS170. There are 269,049 DAM (GATC) sites per haploid genome in C. elegans. Because DALEC captures two tags (in principle) per GATC site, there are a total of 538,098 potential tags (or half sites) per haploid genome. To reduce computation time during alignment of Solexa reads to the genome, each tag was represented by a 16 nucleotide sequence that did not include the 3' GATC.
We excluded DAM tags that occurred more than once in the genome or that mapped to vector or ribosomal sequences. We also excluded tags belonging to two adjacent GATC sites that lie within 20 bp from each other. Under situations where two fully methylated adjacent GATC sites mapped within 20 bp of each other, one site will always be captured at the expense of the other, resulting in undercount of DAM accessibility at such regions. When the distances are slightly above 20 bp, it is conceivable that there may be inherent bias in Mme I sequence preference that leads to the preferential capture of one site over the other, again resulting in undercount. To avoid both situations from skewing our analysis, we excluded such "proximal tags" using the criteria described in Additional File 2, Figure S4). After filtering out proximal, repetitive, and vector/ribosome-derived sequences, we were left with 370,152 in silico tags (per haploid genome) that we could use to align Solexa reads to the genome.
SAGE analysis
SAGE data were obtained from the Genome BC C. elegans Gene Expression Consortium at http://elegans.bcgsc.bc.ca/[43]. We downloaded the March 2006 C. elegans SAGE database using the following relatively standard parameters: Quality filter: 0.99, Hide ambiguous tags: ON, Tag mapping resources: CODING, show only mapped tags: ON, Tags/page: 10, Lowest count cutoff: 1, Hide antisense tags: ON, Remove duplicate ditags: ON, Highest count cutoff: NONE, Sort order: DOWN, Resolve lowest match: ON. We used only long SAGE tags (17 nucleotides long) in our analysis. To determine a total SAGE score for each gene, we collapsed redundant annotations for each gene to a single copy and summed the SAGE score for each annotation. After our filtering criteria, we were left with 13,916 unique genes in our SAGE data set.
All data sets, including raw and aligned Solexa reads, SAGE data sets, in silico generated Dam tags, and gene sets used in our analyses have been deposited into GEO with accession number GSE23042.