Next Generation Sequencing NGS,NGS :,, Massively Parallel Sequencing, NGS Sanger 20 (~600 Gbp vs 3 Mbp) NGS, NGS, DNA RNA, NGS, NGS Illumina (Solexa)

Transcription

1 Next-Generation Sequencing Innovative solutions with SMART technology U TEL (02) TEL (05) TEL (03) TEL (06) TEL (04) TEL (07) 免付費客服專線

2 Next Generation Sequencing NGS,NGS :,, Massively Parallel Sequencing, NGS Sanger 20 (~600 Gbp vs 3 Mbp) NGS, NGS, DNA RNA, NGS, NGS Illumina (Solexa) Roche (454) ABI (SOLiD) Life (Ion Torrent),, : Library Construction Sequencing Analysis Grada A & Weinbrecht K. (2013) J Invest Dermatol 133, e11

3 cdna Synthesis and Library Preparation Kits for Next-Gen Sequencing NGS DNA RNA, NGS RNA microrna TaKaRa Clontech :, SMARTer RNA-Seq Library Construction Kits,!,TaKaRa Clontech! Low Input RNA Cells ( Single Cell) LCM or FFPE samples Complete Transcriptome Coverage Strand Information NGS Fluidigm's C 1 system DNA SMART ChIP-Seq Kit! What is SMART technology? SMART (Switching Mechanism at 5 End of RNA Template) TaKaRa Clontech, sequencing library preparation cdna synthesis kits SMARTScribe RTase, cdna terminal nucleotides, nucleotides Adaptor (SMART Oligo), base-pairing SMARTScribe RTase SMART Oligo 5 Adaptor, Switching Mechanism SMART technology (Full-length gene body coverage) (Sequencing libraries)

4 RNA-Seq Selection Guide Select the right kit for your NGS experiments APPLICATION mrna-seq Total RNA-Seq Total RNA-Seq without strand info with strand info without strand info SAMPLE TYPE ultra low-input total RNA or poly A + RNA intact cells low-input mammalian total RNA high-input mammalian total RNA prokaryotic & non-mammalian rrna-depleted RNA low-input mammalian poly A + RNA LCM, FFPE, or degraded mammalian total RNA prokaryotic & non-mammalian rrna-depleted RNA INPUT AMOUNT (RNA or cells) 10 pg 10 ng 1 1,000 cells Fluidigm C 1 Cell Capture ng 100 ng 1 µg 100 pg 100 ng 100 pg 100 ng ng 200 pg 10 ng rrna REMOVAL not needed not needed RiboGone - Mammalian Kit RiboGone technology built-in not needed RiboGone - Mammalian Kit not needed PLATFORM COMPATIBILITY Illumina Ion Torrent kit included purchase kit separately LIBRARY PREP INCLUDED no no yes yes yes yes no SMARTer Solutions SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing SMARTer Ultra Low RNA Kit for the Fluidigm C 1 System SMARTer Stranded Total RNA Sample Prep Kit - Low Input Mammalian SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian SMARTer Stranded RNA-Seq Kit SMARTer Universal Low Input RNA Library Prep Kit SMARTer Universal Low Input RNA Kit for Sequencing

5 Single Cell Sequencing Single cells, few cells, or ultra-low inputs of total RNA (1 1,000 cells; 10 pg 10 ng) 2013,Single Cell Sequencing Science, Single Cell Sequencing Learn more Single Cell? RNA (Genome Research 2005 Oct; 15(10): ),, Single Cell RNA-Seq? Single Cell RNA-Seq : regulatory pathways 3. DNA Seq Total RNA Seq Single Cell RNA-Seq, Single Cell RNA? RNA, RNA pg RNA! RNA cdna library, RNA! v4 v3 SMART-Seq TM V4 Ultra Low Input RNA Kit for Sequencing SMARTer Ultra TM Low Input RNA Kit for Sequencing-v3 New C 1 SMARTer Ultra TM Low RNA Kit for the Fluidigm C 1 TM System 4

6 v4 New SMART-Seq TM v4 Ultra TM Low Input RNA Kit for Sequencing : SMART-Seq2 locked nucleic acid (LNA) Ultra-low input : 1 1,000 cells 10 pg 10 ng total RNA (RIN>8) NGS : Illumina Ion Torrent NGS :, : A high number of genes identified, full-length transcript data, and minimal rrna reads, RNA-seq data Well established : citations Oligo dt Priming.10 pg sensitivity.work directly with cells.lna technology LNA Clontech ultra low input single cell,,smart- Seq v4! Total RNA Seq SMART-Seq v4! Flowchart of SMART-Seq TM v4 Ultra TM Low Input RNA Kit for Sequencing : SMART-Seq TM v4 Ultra TM Low Input RNA Kit SMART-Seq2 locked nucleic acid (LNA) RNA ds cdna Sequencing DNA Seq 5

7 Sequence Analysis of Ultra-Low Amounts of RNA Ultra Low Samples Small Cell Populations! SMART-Seq v4 RNA 10pg : SMART-Seq v4 Sequencing Metrics DNA Seq Total RNA Seq Sequencing metrics are consistent across RNA input amounts. 10 pg 10 ng of Human Brain Total RNA were used to generate cdna libraries in duplicate with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing. cdna libraries were amplified using 17, 14, 10, or 7 PCR cycles for the 10 pg, 100 pg, 1 ng, or 10 ng libraries, respectively : SMART-Seq v4 Ultra Low Input Kit! Higher sensitivity & Better mappability Higher sensitivity and better mappability with the SMART-Seq v4 kit. Replicate libraries were generated from 10 pg Mouse Brain Total RNA using the SMART Seq v4 kit, the SMARTer Ultra Low v3 kit, or the SMART Seq2 method. 18 PCR cycles were used to amplify cdna libraries with the SMART Seq2 method and SMARTer Ultra Low v3 kit; however, only 17 PCR cycles were needed for the SMART Seq v4 libraries. RNA seq libraries were generated using Nextera XT DNA Library Preparation Kit and sequenced on an Illumina MiSeq instrument. Sequences were analyzed as described in the methods. 6

8 Low expressors Reproducibility is high for lowinput samples. FPKMs from replicate libraries generated from 10 pg of Mouse Brain Total RNA using the SMARTSeq v4 kit, the SMARTer Ultra Low v3 kit, or the SMARTSeq2 method were compared. For transcripts with FPKM <100, the correlation between replicates was much higher for the SMARTSeq v4 kit (Pearson R =0.739; Panel B) compared to the SMARTer Ultra Low v3 (Pearson R = 0.376; Panel A) or the SMARTSeq2 method(pearson R = 0.496; Panel C). For all transcripts (shown in the scatterplots on the right) the correlation between replicates was high for each of the three methods (Pearson R between ), though the SMARTSeq v4 kit did have the highest correlation. Transcripts represented in only one replicate can be seen along the X and Y axes of the scatter plots showing all transcripts. Total RNA Seq Gene body coverage is good for all three library preparation me thods. Gene body coverage shown is the average of two replica te libraries prepared from 10 pg Mouse Brain Total RNA using t he three different cdna synthesis methods. The SMARTer Ultra Low v3 kit produced a slight 3' bias, and the SMART- Seq v4 kit produced a slight 5' bias ; however,the overall covera ge was fairly even. ABRF : Clontech SMARTer Ultra TM Low Input RNA Kit Kits E-f/#.>K@BG4D>6D# %!# $(# $!# (#! W(# DNA Seq ##$###################&#######################%#####################$######################&#####################%#######################%#####################$########################%####################&#################### # 145KB#.3>6C:AR# /43C:6;4# 09XP0# O1###E\# jb39:d#>6#en#c#bq4d#bg:#3>5#%#te-f/u#`b39:d# 0>6WA>F465#E02#]F:6IYABI>6# W$!# 7

9 v3 SMART-Seq Ultra TM Low Input RNA Kit for Sequencing-v3 Ultra-low input : 1 1,000 cells 10 pg 10 ng total RNA (RIN>8) NGS : Illumina Ion Torrent NGS :, : A high number of genes identified, full-length transcript data, and minimal rrna reads, RNA-seq data Well established : citations Oligo dt Priming.10 pg sensitivity.work directly with cells.single tube DNA Seq Total RNA Seq Flowchart of SMARTer Ultra TM Low Input RNA Kit for Sequencing-v3 SMARTer Ultra TM Low Input RNA Kit SMART RNA ds cdna 0.1ng 1ng 10ng RNA Input, (R>0.8) A B C 8

10 Sequence Analysis of Ultra-Low Amounts of RNA SMARTer Ultra Low kit RNA 10pg, Comparison of transcript coverage with different amounts of input RNA. Shown are overlaid plots comparing the average read coverage from libraries made with 1 ng to 0.01 ng (10 pg) of mouse brain total RNA. The x-axis represents gene length normalized to 100%, where 0 is the 5'-end of each transcript and 100 is the 3'-end. The y-axis indicates the average coverage for a set of 724 genes that are moderately to highly expressed in brain tissue. The results are very consistent through the range of input RNA used, with full-length coverage of the transcripts reflecting no systematic 5'- or 3'-bias Illumina Primary Sequencing Metrics Are Similar High mapping to RefSeq High exon: intron ratio Low mapping to rrna Ion Torrent Total RNA Seq Sequencing metrics from single cells. cdna libraries were made from 1 or 1,000 cells (HeLa or Jurkat) using the UL-v3 kit, according to the kit protocol. Illumina library preparation and sequencing was performed as described above. Ion Torrent adapters and barcodes were added using the Ion Xpress Plus Fragment Library Preparation Kit and Ion Xpress Barcode Adapter Kit with 2 ng cdna input. cdna libraries were size selected for 200 base reads and sequenced on an Ion Torrent PGM platform. Gene body coverage from different cell types and input levels. The gene body coverage of the cdna libraries made from 1 or 1,000 HeLa cells (blue and red lines, respectively), or 1 or 1,000 Jurkat cells (green and purple lines, respectively) using the UL-v3 kit was determined using RSeQC. Read coverage was normalized using Excel. DNA Seq 9

11 C 1 SMARTer Ultra TM Low RNA Kit for the Fluidigm C 1 TM System Fluidigm C TM 1 Single-Cell Auto Prep System single cell, SMARTer Ultra TM Low RNA Kit for the Fluidigm C TM 1 System, single cell cdna library! Excellent RNA-seq data (1) (2) 5' or 3' bias (3) <2% rrna (4) Exons (5) High ERCC correlation single cells cdna library mrna-seq, qpcr analysis, Fluidigm DNA Seq Total RNA Seq Single Cell Workflow on C 1 TM System Fluidigm C TM 1 Single-Cell Auto Prep System SMARTer Ultra TM Low RNA Kit cdna library 10

12 Total RNA-seq Low-input (100 pg 100 ng), high-input (100 ng 1 µg), and total RNA of any quality random priming Total RNA-seq? Abundant transcripts ( ribosomal RNA) Non-coding RNA antisense RNA strand of origin Degraded RNA ( FFPE samples RNA) Learn more Clontech SMARTer NGS Kit,! RG RiboGone TM - Mammalian kits Low-input mammalian samples Str SMARTer Stranded RNA-Seq Kit LI SMARTer Stranded Total RNA Sample Prep Kit Low Input Mammalian Total RNA Seq High-input mammalian samples HI SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian Low-input degraded mammalian samples Uni SMARTer Universal Low Input RNA Kit for Sequencing UP SMARTer Universal Low Input RNA Library Prep Kit P Q Low Input Library Prep Kit Library Quantification Kit DNA Seq 11

13 RG RiboGone TM - Mammalian 95% ribosomal RNA ng RNA, FFPE degraded RNA RNA-Seq, rrna removal kit ng (low input ).Short protocol.easy to use The RiboGone TM - Mammalian human, mouse rat ribosomal RNA (rrna) mitochondrial RNA (mtrna) RiboGone RNase H digestion rrna, 5S, 5.8S, 18S, 28S, 12S mtrna RNA (10ng~100ng), 10ng, 100ng, FFPE RiboGone TM - Mammalian, random primer cdna NGS RNA-Seq transcriptome RiboGone RNA, rrna mtrna, NGS,, DNA Seq Total RNA Seq Begin with total RNA of any quality Incubate with RiboGone Hyb Buffer Add provided enzymes and incubate Purify rrna-depleted RNA with SPRI beads Proceed to cdna synthesis using SMART N6 technology RiboGone TM rrna, sequencing! RiboGone - Mammalian removes rrna efficiently from intact and degraded RNA, while retaining noncoding transcripts for analysis. RNA-seq libraries were generated from Human Brain Total RNA (Clontech) or Breast Cancer FFPE RNA (Cureline, extracted using a NucleoSpin totalrna FFPE kit) and treated with the indicated rrna removal method. Reads were mapped to the hg19 genome and read distributions were determined using Picard RNA-Seq Metrics. Libraries generated from RiboGone-treated RNA had comparably low rrna reads to oligo(dt)-enriched RNA (Clontech), while retaining more noncoding reads. 12

14 Str SMARTer Stranded RNA-Seq Kit 99% Strand Identification: strand of origin Broad Input Range: 100 pg ng RNA : : 4 Illumina NGS library :, coding noncoding RNA-Seq Random N6 Priming.100 pg sensitivity.rrna removal kit (RiboGone) required Total RNA Seq Flowchart of SMARTer Stranded RNA-Seq Library Generation Stranded RNA-Seq Kit SMART RNA ds cdna, PCR Illumina adapters, RNA-Seq Library DNA Seq 13

15 Sequence Analysis SMART TM SMART TM RNA (strand identification),smarter Stranded RNA-Seq Kit transcriptome, 5 3, A SMARTer Stranded RNA Seq Kit RNA-Seq library, B C overlapping antisense transcripts DNA Seq Total RNA Seq Human Brain Poly A + RNA (Cat. No ) was spiked with ERCC control RNA and serially diluted to prepare RNA samples containing between 100 pg 100 ng RNA. cdna libraries were prepared using the SMARTer Stranded RNA-Seq Kit according to the kit protocol with twelve different Illumina indices. Libraries were sequenced on an Illumina HiSeq 2000 instrument, with ~300M 2 x 100 bp paired end reads. cdna libraries were mapped against the human genome. (Panel A) The SMARTer Stranded method allowed assignment of sequencing reads to the correct gene in the case of overlapping PHC1 and M6PR transcripts. (Panel B) Strand-specific coverage of the CDR1 locus shows nearly all reads are antisense to the annotated transcript, a finding independently reported elsewhere (2). (Panel C) Comparison of CDR1 gene counts obtained using either a strand-agnostic or strand-aware method. 100 pg RNA 14

16 (Confirmed by ERCC analysis) SMARTer RNA-Seq data MAQC qpcr data Reads mapping to the ERCC data set from the previous experiments were pooled together and the FPKM was plotted against relative transcript abundance. The complete set of 92 ERCC transcripts was identified, with a slope of and R2 of Axes are plotted on a log2 scale. A scatter plot was used to compare differential expression data obtained from SMARTer transcriptome analysis of HURR and HBRR cdna libraries (Table I) and qpcr data for HURR and HBRR from the MAQC project. The slope (0.846) and correlation (0.860) for the comparison line of expression ratio (in RPKM) and qpcr ratio (in Ct) are plotted for HURR and HBRR, in a log2 scale. The transcripts used in this analysis were the 623 of ~900 transcripts present in the MAQC data set that were also detected in both the HURR and HBRR RNA-Seq data sets. Total RNA Seq LI SMARTer Stranded Total RNA Sample Prep Kit-Low Input Mammalian NGS! SMARTer Stranded RNA-Seq Kit + RiboGone TM Mammalian Kit (rrna removal kit) total RNA library! Low-Input ( ng) human, rat or mouse Total RNA Workflow for the SMARTer Stranded Total RNA Sample Prep Kit - Low Input Mammalian. This kit is designed for use with low-input samples containing ng of low-input total RNA samples. cdna libraries generated using this kit are ready for Illumina sequencing and have 99% accuracy in identifying strand of origin information. DNA Seq 15

17 HI SMARTer Stranded Total RNA Sample Prep Kit-HI Mammalian High-Input (100 ng 1 µg) Total RNA 99% Strand Identification : strand of origin RiboGone & SMART : rrna removal, 5 Illumina NGS library : :, coding noncoding RNA-Seq Random N6 Priming.100 ng 1 µg of mammalian total RNA.only ~5 hours Flowchart of SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian library generation. A B C DNA Seq Total RNA Seq Section A. RiboGone technology total RNA samples rrna Section B. SMART technology First-strand cdna synthesis Section C. Template switching, PCR Illumina clustergenerating sequences and indexes 16

18 Sequence Analysis RIN, High-quality libraries Sequence Alignment metrics from RNA of Varying Quality High-quality libraries across varying levels of RNA quality. Libraries were generated from Mouse Liver RNA by chemically shearing until it had a RIN of 3 or 7. Sequencing data showed the percentage of reads that mapped to rrna, exonic regions, intronic regions, intergenic regions, and the correct strand, as defined by Picard analysis. Reproducibility across RNA quality. A scatterplot illustrates the correlations between the FPKMs from two libraries generated from 1 µg Mouse Liver RNA that was chemically sheared until it had a RIN of 3 or 7. MAQC Anaylsis Total RNA Seq MAQC Analysis. RNA-seq libraries were generated with 400 ng of HURR and HUBR. The scatter plot shows the Log2 ratio of FPKMs of HURR/HBRR graphed against the Log2 of the ratio of HURR/ HBRR derived from qpcr Taqman probes. Reproducibility across replicates. RNA-seq libraries were generated from two samples of 100 ng of HURR. The scatterplot illustrates correlations between the FPKMs (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) from the two libraries. ERCC transcripts with correct strand 99% Sequence Alignment metrics Sequence Alignment Metrics. 400 ng of HURR and HBRR with ERCC Spike-In RNA were treated with this kit. Alignment data is displayed for both libraries, with the percentage of reads that mapped to rrna, exonic regions, intronic regions, intergenic regions, and the correct strand, as defined by Picard analysis. DNA Seq 17

19 Uni SMARTer Universal Low Input RNA Kit for Sequencing FFPE laser-captured Degraded (RIN 2-3) non-polyadenylated RNA library Low-Input Sample : 200 pg rrna-depleted RNA / 2 ng total RNA (Complete Transcriptome Coverage), 5' or 3' bias Illumina Ion Torrent NGS Random N6 Priming.200 pg sensitivity.rrna removal kit (RiboGone) required Flowchart of SMARTer Universal Low Input RNA Kit 1 First strand systhesis and tailing by SMARTScribe TM Reverse Transcriptase DNA Seq Total RNA Seq Template switching and extension by SMARTScribe TM Reverse Transcriptase cdna amplification Adapter removal 18

20 Sequence Analysis RNA-seq data Mappability > 80% Number of genes > 15,000 Total RNA Seq RNA Input, (Reproducibility >0.98) A B C D E F Scatter plots of expression (RPKM) for cdna libraries prepared from rrna-depleted Human Brain Total RNA. Panels A C. Comparisons of pairs of cdna library replicas (Replica #1 and Replica #2) created from 156 pg, 780 pg, and 7.8 ng of input RNA show high reproducibility across a wide range of RNA concentrations. Panels D F. Comparisons of cdna libraries generated from pairs of RNA input amounts (780 pg and 156 pg, 7.8 ng and 156 pg, and 7.8 ng and 780 pg) show a high correlation, suggesting consistency across input levels. Axes are plotted on a log10 scale. Insets indicate the co-efficient of correlation by Spearman analysis (ρ) and by Pearson correlation (R) DNA Seq 19

21 Sequence Analysis (Confirmed by ERCC analysis) DNA Seq Total RNA Seq qpcr The ERCC (External RNA Control Consortium) Spike-In Mix (92 transcripts) was chemically sheared and 4 microliters (1:1,000 dilution) was spiked into 100 ng of chemically sheared Human Brain Total RNA prior to rrna depletion. Two cdna library replicates were generated with each of 156 pg, 780 pg, and 7.8 ng of rrna-depleted RNA/ERCC mix using the SMARTer Universal Low Input RNA Kit for Sequencing. RPKM of the ERCC transcripts was plotted against the attomoles per transcript spiked in. The slope, R2, and the number of transcripts are indicated per replicate. Pearson correlation (R) values per replica set were determined to be 0.998, 0.999, and for Panels A C, respectively. Axes are plotted on a log2 scale. Scatter plots were used to compare differential expression data obtained by sequencing cdna libraries created with the SMARTer Universal RNA Kit [106 pg HURR/159 pg HBRR (A), 530 pg HURR/790 pg HBRR (B), and 5.3 ng HURR/7.9 ng HBRR (C)] and quantitative PCR (qpcr) data available for HURR and HBRR through the MAQC (MicroArray Quality Control) project (2). The slope and correlation (R) for the comparison line of expression ratio (in RPKM) and qpcr ratio (in Ct) are plotted for HURR and HBRR, in a log2 scale. The number of genes present in both data sets is indicated. HURR = Human Universal Reference RNA. HBRR = Human Brain Reference RNA. 20

22 P Low Input Library Prep Kit SMARTer Universal Low Input RNA Kit for Sequencing SMARTer Ultra Low RNA Kit for Illumina Sequencing-v3 dsdna Preparation,, 2 50 pg- 20 ng of starting dsdna fragmented dsdna Illumina sequencing-ready libraries multiplexed libraries, high throughput sequencing! Highly sensitive, simple and fast protocol.single-read or paired-end sequencing The Low Input Library Prep Kit workflow 2 hr OK! Total RNA Seq UP SMARTer Universal Low Input RNA Library Prep Kit Library, SMARTer Universal Low Input RNA Kit Low Input Library Prep Kit DNA Seq 21

23 Q Library Quantification Kit Highly sensitive : qpcr-based, libraries Robust :, 30%- 85% GC content genomic libraries Accurate : control, library Complete kit : qpcr primers reagents DNA standards qpcr based method of DNA library quantification Library? library, loading, NGS data! cdna adapters cdna low cluster density (= poor quality data) DNA Seq Total RNA Seq SMARTer Workflow Library Quantification Kit Workflow Step 1 Library dilution Step 2 Reaction set-up Step 3 Obtaining results 1. Prepare qpcr/primer mix 1. Prepare qpcr Plate 1. Analyze data 2. Make 1:1000 dilution of dsdna library 2. Run qpcr protocol 2. Calculate library concentration 22

24 ChIP-seq Single-stranded or double-stranded, low-input (100 pg 10 ng) ChIP DNA Learn more ChIP experiments low input sample library? ChIP DNA, ChIP-seq library ligation, DNA SMART TM ChIP-Seq kits SMART (ligation-free manner) ChIP-Seq libraries,! ChIP ChIP Elute Kit DNA SMART TM ChIP-Seq Kit ChIP Elute Kit ChIP assay DNA 1 ssdna ( overnight!) ChIP assay protocol DNA SMART ChIP-Seq Kit qpcr applications Total RNA Seq DNA SMART TM ChIP-Seq Kit Ligation-independent : SMARTer Illumina -specific sequencing adapters ChIP-seq library Low Input Compatibility : 100 pg 10 ng ssdna dsdna, total DNA from 10,000 1,000,000 cell ChIP experiments Save Your Time : 4!! Single Tube Workflow ChIP Elute Kit DNA Seq DNA SMART TM ChIP-Seq Kit 23

25 ChIP Elute Kit DNA SMART ChIP Seq Kit ChIP assay protocol Total time ~1 hour ChIP Elute Kit DNA Seq Total RNA Seq 5 OK! DNA SMART ChIP Seq Kit Total time ~4 hour DNA elution, Cross-linking reversal, DNA purification and concentration DNA library synthesis! 24

26 , ChIP Elute Kit (1 hr) ChIP- seq libraries Sequencing metrics comparing ChIP seq libraries generated with ChIP Elute or traditional cross linking reversal methods. Across all sequencing metrics, ChIP DNA produced using the ChIP Elute Kit generated ChIP seq libraries that were comparable to those produced using traditional, slow cross linking reversal methods. Total RNA Seq The shape and location of peaks are similar for different cross linking reversal and elution methods and across input amounts. ChIP seq libraries made from DNA eluted with either the ChIP Elute Kit or traditional method had high amounts of overlap in the number of peaks identified. The percent overlap of peaks identified in ChIP seq libraries preparedfrom DNA isolated with the ChIP Elute and traditional method were 90% and 89% for 1 ng and 250 pg samples, respectively. DNA Seq 25

27 Sequence Analysis >90% mapping of reads Input, No. of peaks identified Sequencing metrics from various amounts of input DNA Inputs >0.5 ng, Non-redundant rate ENCODE project 0.8 peaks, ENCODE data DNA Seq Total RNA Seq D Libraries generated with the DNA SMART ChIP-Seq Kit have high reproducibility. The reproducibility between technical replicates was similar across input amounts (Panel A). The non-redundant rate (normalized for 10 million uniquely mapped reads) was well above the standard recommended by the ENCODE project (0.8) for inputs >0.5 ng (Panel B; error bars indicate the standard deviation of two technical replicates). Compared to the 4 ng library, the number of peaks were similar across lower input libraries (Panel C). The shape and location of the peaks was similar across input levels, and matched very well to ENCODE data (293 cells, anti-h3k4me3 antibody, U. Washington), even for as little as 50 pg input DNA (Panel D). 26

28 Total (unquantified) ChIP DNA DNA SMART TM ChIP-Seq Kit library, ChIP-seq data Sequencing metrics from total DNA from specified numbers of cells Inputs,! peaks, ENCODE data DNA SMART ChIP-Seq Kit libraries maintain consistent representation of sequences even at the lowest input levels. Peaks from different numbers of cells were identified using 6 million (for 10,000 cells) or 9 10 million (for 50,000 1,000,000 cells) uniquely mapped reads. There was a high amount of overlap across the number of input cells (Panel A). The peaks were of similar shape across cell inputs and matched the peaks obtained by the ENCODE project (293 cells, anti-h3k4me3 antibody, U. Washington; Panel B). Additionally, the total number of peaks overlapping with those identified in the ENCODE project was high (Panel C). Total RNA Seq DNA Seq 27

29 PRODUCTS Cat. # Product Size Single-Cell mrna-seq SMART-Seq TM v4 Ultra TM Low Input RNA Kit for Sequencing Rxns SMARTer Ultra TM Low Input RNA Kit for Sequencing - v Rxns mrna-seq with the Fluidigm C 1 System SMARTer Ultra TM Low RNA Kit for the Fluidigm C TM 1 System, 2 IFCs Each SMARTer Ultra TM Low RNA Kit for the Fluidigm C TM 1 System, 10 IFCs Each Total RNA-Seq Low-input total RNA-Seq RiboGone TM - Mammalian 6 Rxns RiboGone TM - Mammalian 24 Rxns SMARTer Stranded RNA-Seq Kit Rxns SMARTer Stranded Total RNA Sample Prep Kit - Low Input Mammalian 24 Rxns SMARTer Stranded RNA-Seq Kit HT 96 Rxns SMARTer Universal Low Input RNA Kit for Sequencing 10 Rxns SMARTer Universal Low Input RNA Kit for Sequencing 25 Rxns SMARTer Universal Low Input RNA Library Prep Kit 10 Rxns SMARTer Universal Low Input RNA Library Prep Kit 25 Rxns High-input total RNA-Seq SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian Rxns Low-input ChIP-Seq ChIP Elute Kit 50 Rxns DNA SMART TM ChIP-Seq Kit Rxns Accessory Products for Illumina Sequencing Low Input Library Prep Kit 12 Rxns Library Quantification Kit 500 Rxns DNA Standards for Library Quantification 50 Rxns

30 TEL (02) TEL (05) TEL (03) TEL (06) TEL (04) TEL (07) 免付費客服專線