TRUSTED BRAND Infinium and GoldenGate assay systems SureSelect Methyl-seq Target Enrichment System MassARRAY EpiTYPER NimbleGen SeqCap Epi Enrichment

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1 We are TWINS!! Epigenetics Secret Garden Why are we so different? TEL (02) TEL (05) TEL (03) TEL (06) TEL (04) TEL (07) 免付費客服專線

2 TRUSTED BRAND Infinium and GoldenGate assay systems SureSelect Methyl-seq Target Enrichment System MassARRAY EpiTYPER NimbleGen SeqCap Epi Enrichment System Quick Guide of Popular Epigenetics Products 02

3 Epigenetics Secret Garden Bisulfite Conversion-The Gold Standard EZ DNA Methylation Kit DNA Input:100 pg - 2 µg (optimal input : ng) : Direct Lightning, bisulfite-converted DNA PCR MSP array NGS RNA Methylation RNA methylation # R5001/R5002 bisulfite conversion, non-methylated cytosine uracil; DNA PCR,uracil thymine, CT conversion Selection Guide EZ DNA Methylation Kit EZ DNA Methylation-Gold Kit EZ DNA Methylation-Lightning Kit EZ DNA Methylation-Direct Kit CT conversion DNA D Rxns D Rxns 500 pg-2 ug DNA > 99% > 80% hrs D Rxns D Rxns 500 pg-2 ug DNA > 99% > 75% 3 hrs D Rxns D Rxns 100 pg-2 ug DNA > 99.5% > 80% 1.5 hrs D Rxns DNA( 50 pg), cells( 10), blood, > 99.5% > 80% 4 hrs D Rxns tissue, FFPE 03

4 Sensitive 5-mC and 5-hmC Quantification 5-mC and 5-hmC ELISA Kits ELISA 5-mC 5-hmC, : 0.5% 5-mC 0.02% 5-hmC per 100 ng input DNA 5-mC ELISA/5-hmC ELISA DNA 5-mC/5-hmC, mass spectrometry (MS) The 5-mC DNA ELISA Kit can quantify 5-mC in numerous DNA samples with close correlation to LC-MS analysis. Percent 5-mC was calculated for the following genomic DNA samples: human brain (HB), human kidney (HK), human embryonic stem cell (HESC), mouse brain (MB), mouse kidney (MK), and mouse testes (MT). The percent 5-mC detected in DNA samples by 5-mC DNA ELISA Kit (ELISA) strongly correlates to mass spectrometry (MS) data of 5-mC found in the respective gdna sample. 5-hmC Quantification. Percent 5-hmC in mammalian DNA samples quantified by mass spectrometry or Quest 5-hmC ELISA Kit. Inlaid image represents relative amounts of 5-hmC in triplicate gdna samples. 5-mC DNA ELISA Kit D x 96-well D x 96-well Quest 5-hmC DNA ELISA Kit D x 96-well D x 96-well 04

5 Epigenetics Secret Garden Enrichment of 5-mC -containing DNA Methylated-DNA IP Kit anti-5-methylcytosine monoclonal antibody DNA control DNA primers, DNA PCR whole-genome amplification ultra-deep sequencing microarray IP methylated DNA Salmon sperm genomic DNA "spiked" with non-methylated/methylated control DNA was processed with the kit and the eluted DNA was amplified by PCR using the supplied control primers. Digestion of the amplicons with Nco I produced two 175 bp fragments for the methylated DNA control or one 350 bp fragment for the nonmethylated DNA control. Methylated-DNA IP Kit D Rxns 05

Post-Bisulfite Library Preparation Pico Methyl-Seq Library Prep Kit :FFPE ssdna, 10 pg DNA random primer bisulfite treatment DNA PCR, WGBS (Whole Genome Bisulfite Sequencing) library, bisulfite

6 Post-Bisulfite Library Preparation Pico Methyl-Seq Library Prep Kit :FFPE ssdna, 10 pg DNA random primer bisulfite treatment DNA PCR, WGBS (Whole Genome Bisulfite Sequencing) library, bisulfite conversion library preparation,ligation-free gel-free, Illumina's sequencing platform : HiSeq MiSeq (A) Electropherogram of a Pico Methyl-Seq (WGBS) library prepared using 100 pg of human gdna. (B) Gel of libraries prepared from varied amounts of human gdna. Fragments ranged between 150 and 350 bp. Pico Methyl-Seq Library Prep Kit D Preps Pico Methyl-Seq Library Prep Kit D Preps 06

7 active DNA demethylation pathways. Previous studies amplific demonstrate that it plays a role in cell differentiation and Epigenetics Secret concent Garden carcinogenesis: Cells that are more stem- and progenitor-like have greatly reduced levels of 5-hmC compared with more (a) 5-hmC Library Preparation differentiated RRHP cells. Similarly, tumor cells display less 5-hmC than 5-hmC their normal Library counterparts Prep independent Kit of either grade or stage, suggesting that global loss of 5-hmC may be an early Msp event I, in carcinogenesis. glucosylated-5-hmc Several methods, have glucosylated-5-hmc been described Msp I, to profile 5-hmC library at the genomic level: Most are enrichmentbased ng low via input immunoprecipitation DNA or other bioorthogonal labeling 100 schemes, strand-specific mapping several of conversion 5-hmC DNA methods have also been Illumina's described sequencing that exploit platform selective : HiSeq oxidation. MiSeq Here we employ a new method which combines modification-sensitive restriction enzymes with next-generation sequencing approaches to allow genome-wide 5-hmC mapping at single-site resolution in several (b) β-glucosyltransferase(β-gt) families of carcinomas. This 5hmCnew, method should provide Msp I a unique tool in enhancing our understanding of the interplay of genetic and epigenetic regulations in carcinogenesis. Methodology (a) (c) (b) Figure cancero conten and no Region concent that tu librarie 5-hmC This re profoun (c) D preps RRHP 5-hmC Library Prep Kit D preps (d) Total read Mapped re Mappabili # of tagged r tagged 07 read

Region Specific 5-mC Analysis OneStep qmethyl MSREs (Methylation Sensitive Restriction Enzymes), CpG non-methylated CpG:MSREs dsdna,qpcr

primers, Specifications Format... 96-Well Plate DNA Input...20 ng in 5 µl Processing Time.

sample 2 DNA MSREs digestion, test reaction; DNA MSREs digestion, reference reaction 3 primer set, qpcr ( SYBR green Taqman probe ) 4

8 Region Specific 5-mC Analysis OneStep qmethyl MSREs (Methylation Sensitive Restriction Enzymes), CpG non-methylated CpG:MSREs dsdna,qpcr Ct reference methylated CpG:MSREs dsdna,qpcr Ct reference bisulfite conversion, qpcr DNA single-, multi-locus DNA methylation control DNA primers, Specifications Format Well Plate DNA Input...20 ng in 5 µl Processing Time...~4 hours Add DNA Real-time PCR Simple, One Step 4 hr, DNA Non-methylated DNA Input Methylated DNA Input 1 non-methylayed methylayed DNA sample 2 DNA MSREs digestion, test reaction; DNA MSREs digestion, reference reaction 3 primer set, qpcr ( SYBR green Taqman probe ) 4 non-methylated DNA: Ct Test > Ct Ref methylated DNA: Ct Test Ct Ref OneStep qmethy (with SYTO 9 dye for qpcr) D x 96 well OneStep qmethyl -Lite (without SYTO 9 dye) D x 96 well 08

9 Epigenetics Secret Garden Chromatin Analysis Zymo-Spin ChIP kit, ChIP ChIP DNA 6 µl ChIP DNA ChIP-qPCR, ChIP-Seq ChIP DNA ENCODE Quality ChIP Workflow : Browser tracks depicting H3K4me3 ChIP-Seq assay using the Zymo-Spin ChIP Kit. Peaks overlap the same sites identified at the Broad Institute of MIT and Harvard as part of the ENCODE project. ChIP DNA Clean & Concentrator, 2 ChIP sample DNA DNA, 6 µl ChIP DNA, PCR, arrays, sequencing ChIP DNA ChIP DNA Purification Comparison : ChIP assays were performed with HeLa cells using ChIP-grade anti-h3k4me3 and rabbit IgG antibodies. Both total and immunoprecipitated chromatin were reverse cross-linked and recovered using either the ChIP DNA Clean & Concentrator (included in the Zymo-Spin ChIP Kit), DNA recovery kit from Supplier Q, Chelex -100 protocol or phenolchloroform extraction. The amount of ChIP DNA was determined using qpcr with primers specific to the GAPDH promoter. ChIP DNA enrichment is graphed as % input. Zymo-Spin ChIP Kit D preps D preps ChIP DNA Clean & Concentrator Kit (Uncapped) D Preps ChIP DNA Clean & Concentrator (Capped) D Preps 09

Accurately Quantitate DNA Methylation Epigenetic Standards non-methylated methylated human DNA negative positive control bisulfite conversion (primer design, bisulfite PCR and sequencing reactions ),

Sample 2 is positive for a Non-Methylated Template and Sample 3 contains Methylated and Non-Methylated Templates.

10 Accurately Quantitate DNA Methylation Epigenetic Standards non-methylated methylated human DNA negative positive control bisulfite conversion (primer design, bisulfite PCR and sequencing reactions ), non-methylated methylated human DNA, calibration curves DNA bisulfite primer Example MSP experiment using MSP designed primers for RASSF1. Sample 1 is positive for a Methylated Template. Sample 2 is positive for a Non-Methylated Template and Sample 3 contains Methylated and Non-Methylated Templates. MSP experiment also shows proper controls: Meth (+) DNA Control D Human Methylated DNA, Meth (-) DNA Control D Human Non-methylated DNA. 2% Agarose Gel, 130V for 35 mins. M = Methylated Template, U = Non-Methylated Template Gel electrophoresis depicting genomic DNA, bisulfite converted genomic DNA and genomic DNA amplified with bisulfite-specific primers. Lane 1 Input DNA: Universal Methylated Human DNA Standard (D5011). Lane 2- Bisulfite converted Universal Methylated Human DNA (D5011) using EZ-DNA Methylation Direct (D5020). Lane 3 Universal Methylated Human DNA (D5011) bisulfite converted and amplified with supplied hmlh1 control primers. Human Methylated & Non-Methylated (WGA) DNA Set (DNA w/ primers) D set Human Methylated & Non-Methylated DNA Set (DNA w/ primers) D set Universal Methylated Human DNA Standard D Rxns Universal Methylated Mouse DNA Standard D Rxns 10

Epigenetics Secret Garden Enzymes ZymoTaq DNA Polymerase Hot-start, bisulfite-converted DNA PCR ZymoTaq PCR PCR products of immunoprecipitated, methylated DNA vary depending on the hot-start

11 Epigenetics Secret Garden Enzymes ZymoTaq DNA Polymerase Hot-start, bisulfite-converted DNA PCR ZymoTaq PCR PCR products of immunoprecipitated, methylated DNA vary depending on the hot-start polymerase used. dsdna Shearase Plus dsdna enzyme DNA library construction, Next-Generation sequencing methylated DNA immunoprecipitation (MeDIP) DNA Degradase & DNA Degradase Plus, DNA Degradase DNA nucleotide DNA Degradase Plus DNA nucleoside HPLC, TLC, LC-MS ZymoTaq Polymerase E Rxns E U DNA Degradase E Rxns E U dsdna Shearase Plus E U E U DNA Degradase Plus E U E U 11

12 Epigenetics Secret Garden TEL (02) TEL (05) TEL (03) TEL (06) TEL (04) TEL (07) 免付費客服專線 U/A/UV070117

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/ DNA DNA Clean & Concentrator DCC 讓您笑嘻嘻! DM TEL (02)2720-2215 TEL (05)2844-162 TEL (03)6684-5 8 6 TEL (06)2890-665 TEL (04)2463-3591 TEL (07)3470-143 免付費客服專線讓您笑嘻嘻!... 1 Zymo Clean-Spin Technology spin