Add 5µl of 3N NaOH to DNA sample (final concentration 0.3N NaOH).

Transcription

1 Bisulfite Treatment of DNA Dilute DNA sample to 2µg DNA in 50µl ddh 2 O. Add 5µl of 3N NaOH to DNA sample (final concentration 0.3N NaOH). Incubate in a 37ºC water bath for 30 minutes. To 55µl samples add: 510µl 3.9M sodium bisulfite (ph 5.0)(3.3M final) 30µl 10mM hydroquinone (0.5mM final) 15µl H 2 O Incubate overnight (10-14 hours) at 55ºC protecting from light. Implement Wizard DNA Resin Based Clean-Up System (Promega Cat. #A7280) Sequencing ABI BigDye Terminator v1.1 Cycle Sequencing Kit was used according to the manufacturer s instructions with the following alterations: For direct sequencing of PCR products, the reaction mix was diluted 1:2. For sequencing of cloned PCR products, the reaction mix was diluted 1:8. In both cases reaction buffer was added to make up for reduced reaction mix components.

2 PCR of Bisulfite DNA Primer List Primer Name Primer Sequence Ann Ext Cyc CpG1-N-for GTT GTT TAG GGT TAT TTT TTT T CpG1-N-rev TAA ATA AAA TTT TTT TAA A CpG1-O-for GAG TAT TAT TAG TGT TTG TAA T CpG1-O-rev AAT TTT TTA AAT TTT ATA AT CpG1.5-N-for GGG TGG TGT TTG GTA TAG GTT G CpG1.5-N-rev CTA AAA ATA CTT TCA ACT AAA A CpG1.5-O-for GAG TTT TTT TTA TTT ATT ATG GG CpG1.5-O-rev AAC TAC TCT AAA CAA ATA C CpG3A-N-for AAT ATA TGG TTT TTG TTA TA CpG3A-N-rev CAC CAA AAA AAA ATA AAA TAC AAT CAC CpG3A-O-for GTT GGT TAT AAT TAA AAT AAA TTA G CpG3A-O-rev ATA AAA CTA CAA ATT TCA TAT AAC AAC CpG3B-N-for GTT GTT ATA TGA AAT TTG TAG TTT TAT CpG3B-N-rev CCT ACC TTA AAA ATT AAC CCA AAA TCC CpG3B-O-for GGT GAT TGT ATT TTA TTT TTT TTT GGT CpG3B-O-rev CCT TAA ACT AAA AAC TA CAA AAA CTA A CpG6-N-for GGG ATG TTT TTT TTA AAT TTA G CpG6-N-rev CCT TTA TTA ATT TTC TTC ATT TTC CpG6-O-for GGT TAG AGA TTT TTA AGA GG CpG6-O-rev TAA ACC CCT AAA AAA CTC ACT C Table conventions: All sequences are presented 5 to 3. O-Outside Primer, N-Nested Primer, for-forward Primer, rev-reverse Primer, Ann-Annealing Temperature ( C), Ext- Extension Time (sec.), Cyc-Total Cycles. PCR was performed using the above specifications in the following protocol PCR Reactions (25µl for outside PCR, 50µl for nested PCR) 10 X Reaction Buffer 1 X Primers (for & rev) 2 µm dntp mix 100 nm Taq Enzyme 1unit / 25 µl Template (outside) 100 ng bisulfite treated DNA Template (nested) 5 µl outside PCR PCR programs all started with 3 minute initial denaturation at 94 C. Cycle cores included 30 seconds of 94 C denaturation, variable annealing temperatures for 30 seconds, and 72 C extension cycles for variable times. A final extension of 72 C for 10 minutes ends the programs.

3 Mathematics for Calculating Relative Methylation ABI sequence data, in the form of histograms, were exported as TIFF files. Histograms were imported into Scion Image software v4.0.2 (Beta). Area under the curve (AUC) analysis was performed by tracing histogram peeks and determining the number of pixels under Cytosine/Thymidine curves at each CpG residue. Additionally, AUC analysis was performed on two converted non-cpg Cytosine residues. These residues were used to determine background *. Area under CpG Cytosine = aucc Area under CpG Thymidine = auct % conversion = (auct / (auct + aucc))*100 background (or % not converted) = % conversion two separate background measurements for each sequence was taken and averaged. correction = (background / 100) * (aucc + auct) Methylation = ((aucc correction) / (auc C correction + auct)) *100 * Background arises from the incomplete nature of the bisulfite reaction. A certain percentage of what appears to be methylated Cytosines are actually just unmethylated cytosines that failed to be converted by the bisulfite reaction. The conversion failure rate at any given CpG Cytosine should be equal to the conversion failure rate at any

4 given non-cpg Cytosine. The raw data for background determination were the aucc and auct at two separate non-cpg Cytosine residues for each sequence. Sample of Bisulfite Sequencing Peak Measurements Sample Sequence from CpG 1 MEF Mll+/+ Cells Sample Sequence from CpG 1 MEF Mll-/- Cells CpG Cytosine Peaks are Measured CpG CYtosine Peaks are Measured CpG Thymidine Peaks are Measured CpG Thymidine Peaks are Measured

5 RT-PCR All RNAs were isolated using Tri-Reagent (Sigma #T9424) according to the manufacturer s protocol. RNA was treated with DNA-free (Ambion #1906) for remove any genomic contamination. Complimentary DNA was synthesized with SuperScript First-Strand Synthesis System (Invitrogen #12371). All PCR reactions were performed using the following protocol: 1 cycle of 94 C for 2 min, multiple cycles (depending on the gene of interest) of 94 C for 1 min., 60 C for 1 min. and 72 C for 1 min., followed by 1 cycle of 72 C for 10 min. PCR products were analyzed with ethidium bromide by electrophoresis on a 2% agarose gel. Primers used are as follows (both forward and reverse primers presented 5 to 3 ): amplicon Forward Primer Reverse Primer gapdh GTG AGG CCG GTG CTG AGT AT TCA TGA GCC CTT CCA CAA TG AB transcript CGG TGA TTT AGG TAG TTT CCT GTT G CAC AGC GCC GAG GAA GAC Hoxa9 TGA GAG CGG CGG AGA CA TGC CTT TCG GTG AGG TTG AG Mll TCA GGC AGA CAA GCT TCC AA CAC TCA AGG TGG GCA TGT CA Real-Time RT-PCR All RNAs were isolated using Tri-Reagent (Sigma #T9424) according to the manufacturer s protocol. RNA was treated with DNA-free (Ambion #1906) for remove any genomic contamination. Complimentary DNA was synthesized with SuperScript First-Strand Synthesis System (Invitrogen #12371). cdna was analyzed using ABI Prism 7300 sequence detector. All reactions were performed in triplicate and gene expression levels detected using SYBR Green reagents (Bio Rad # ). Fold difference in gene expression was calculated using 2 - Ct method.