转录组合并数据分析报告-454

上海众信生物技术有限公司

数据分析报告

2013年5月1日

XX转录组合并数据分析报告

目 录

1．分析要求 ....................................................................................................................................................3 2．分析流程及结果.......................................................................................................................................3 2.1 实验流程 ...............................................................................................................................................3 2.2 数据分析流程 ......................................................................................................................................4 2.3 原始数据 ...............................................................................................................................................4 2.4 序列拼接 ...............................................................................................................................................5 2.5 SSR分析................................................................................................................................................6 2.6 SNP分析 ...............................................................................................................................................6 2.7 BLAST注释.............................................................................................................................................7 2.8 GO分类 .................................................................................................................................................7 2.9 差异基因分析 ......................................................................................................................................9 2.10 UNIGENE比对 ....................................................................................................................................9 3．结果文件及说明.....................................................................................................................................10 3.1 软件参数设置 ....................................................................................................................................10 3.2 结果文件目录说明 ...........................................................................................................................11 4. 参考文献 ...................................................................................................................................................12 5. 附录（实验部分PROTOCOL） .......................................................................................................12 5.1 RNA提取 ............................................................................................................................................12 5.1.1 RNA precipitation .......................................................................................................................12 5.1.2 RNA wash.....................................................................................................................................13 5.1.3 RNA resuspension .......................................................................................................................13

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5.2反转录成CDNA ................................................................................................................................13 5.2.1 First-strand cDNA synthesis, set up 2 tubes for 2ug polyA RNA, for each tube: ............14 5.2.2 Second strand synthesis, for each tube:..................................................................................14 5.2.3 Phenol chloroform (Corrosive) isoamyl alcohol (PCI) and EtOH precipitation ............15 5.2.4 Gel separation.............................................................................................................................16 5.2.5 Gel extraction..............................................................................................................................16 5.2.6 Sonication ....................................................................................................................................17 5.3 454 LIBRARY PREPARATION ................................................................................................................17 5.3.1 Fragmentation of RNA...............................................................................................................17 5.3.2 Double-stranded cDNA Synthesis............................................................................................18 5.3.3 Fragment End Repair ................................................................................................................19 5.3.4 AMPure Bead Preparation........................................................................................................20 5.3.5 Adaptor Ligation.........................................................................................................................20 5.3.6 Small Fragment Removal ..........................................................................................................21 5.3.7 Library Quantitation ..................................................................................................................21 5.3.8 cDNA Library Quality Assessment ..........................................................................................23 5.3.9 Preparing Working Aliquots .....................................................................................................23

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XX转录组合并数据分析报告

1．分析要求

对经预处理的四个组织的454测序结果进行合并拼接、以及对此拼接结果进行注释、SNP分析、SSR分析、GO功能分类等分析。

2．分析流程及结果

2.1 实验流程

RNA提取 反转录成cDNA 454测序 454 Library制备

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2.2 数据分析流程

2.3 原始数据

实验共得到4个组织各1/2 run数据，合计2个run的测序数据。数据预处理已在上次报告中给出，这里对经过预处理后合并的数据进行reads长度频数统计，详细结果见reads_statistics.xls中original_reads_stat sheet。图2.3.1为reads长度分布示意图，表2.3.1给出预处理后reads基本统计信息。

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图2.3.1 reads长度分布示意图

表2.3.1 预处理后reads基本统计信息

ALL Reads num 2348,945 Total bases 699,966,000 Length range(bp) 100-627 Average length(bp) 298 Length std 90.9 2.4 序列拼接

应用Newbler v2.5.3软件进行序列拼接，参数如3.1-d所示，拼接结果统计如表2.4.1所示，拼接结果在每个组织的分布情况如表2.4.2所示。

表2.4.1 拼接结果统计

Isotig num 84,867 Isotig bases 41,802,506 N50(bp) 547 Average length(bp) 492.6 Average depth 27.3 Singlets num 42,844 表2.4.2 拼接结果各组织分布统计

Tissue name ji shen gan

Singlets num 5745 9769 5841 Singlets rate (%) 13 23 13 Isotig num 30959 13463 7932 Isotigs rate (%) 36 16 9 5

nao 21489 51 32813 39 在Newbler软件中引入Isotig的概念。从结构上讲可把contig比作exon，把Isotig比作整个转录本，不同的Isotig可由1至多个contig进行不同组合而成。Isotig更能反映转录本的真实情况，后续分析将使用Isotig及singlets序列进行。

2.5 SSR分析

将2.4得到的Isotig 及 Singlets用MISA软件进行SSR分析，参数如3.1-e所示。统计结果如表2.5.1所示，详细结果见结果文件。

表2.5.1 SSR分析结果统计

SSR type statistics 含有SSR的序列个数 2个以上SSR序列个数 混合型SSR个数 单碱基型SSR个数 双碱基型SSR个数 3碱基型SSR个数 4碱基型SSR个数 5碱基型SSR个数 6碱基型SSR个数 1-6碱基型SSR总数 All 17082 2060 1385 5787 8406 2032 760 87 10 14581 2.6 SNP分析

将2.4得到的Isotig序列进行SNP分析。用ssahasnp软件将每个Isotig所对应的reads比对到Isotig，通过拼接时覆盖到同一位置上碱基的不同来预测可能的SNP位点，其中SNP覆盖率大于10以及突变率大于20%，详细结果见SNP/all_SNP_result.xlsx文件。对SNP类型进行统计，统计图如图2.6.1所示。不同组织SNP统计结果见结果文件。

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4004201019941780626480206468overall transitionsA<->GC<->Toverall transversionA<->T图2.6.1 SNP类型统计

G<->TC<->GA<->C

2.7 blast注释

对2.4得到的Isotigs及singlets进行blastx注释。与Swiss-Prot数据库进行比对，合并比对结果，取e值小于等于1e-3，alignment大于等于30%的比对结果。详细结果见结果文件。

2.8 GO分类

根据2.7的比对结果进行GO分类，得到所有序列在Gene Ontology的三大分类：molecular function, cellular component, biological process的各个层次上的分布情况。详细结果见结果文件。并给出各分类第二层次上的分布图形如图2.8.1，图2.8.2，图2.8.3所示。

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图2.8.1 biological process level 2 distribution

图2.8.2 cellular component level 2 distribution

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图2.8.3 Molecular function level 2 distribution

2.9 差异基因分析

将参与拼接的四个组织reads分布情况进行统计，然后分别将每个组织两两比较，得出两者之间的差异基因以及无差异基因。这里采用Fisher检验，参考表达量为各组织表达量总和，差异基因阈值P：0.05，详细结果见：ji_nao，ji_gan，ji_shen，nao_gan，nao_shen，gan_shen这六个表，其中带颜色标记的差异基因：红色代表前者表达量大于后者，例如ji_shen，则为ji表达量大于shen表达量；绿色表达前者表达量小于后者，例如ji_shen，则为ji表达量小于shen表达量；黑色字体即无颜色表示为无差异基因。

2.10 UniGene比对

应用tblastx程序将拼接出的Unigene（127711）与指定的7种鱼的EST/mRNA序列进行比对，根据如下参数提取比对结果：alignment >= 40% (query or subject); E: 1e-05。比对统计结果如表2.10.1所示，详细结果见结果文件。

表2.10.1 比对统计结果

Species of EST/mRNA Carassius auratus Cyprinus carpio

Unique num of unigene align to EST/mRNA 5298 8895 Percent % 4.15 6.96 9

Danio rerio Fugu rubripes Gasterosteus aculeatus Oryzias latipes Tetraodon nigroviridis Total unique 17377 2411 7747 8037 3674 24092 13.61 1.89 6.07 6.29 2.88 18.86 3．结果文件及说明

3.1 软件参数设置

以下仅列出分析所涉及到的软件参数设置，自写程序由于不涉及到任何参数及版权问题故不公开。

a. Seqclean (默认参数)

b. Lucy：-m 50 –e 0.03 0.03 –w 30 0.03 10 0.1 –b 4 0.03

参数 -m -e[1] -e[2] -w[1] -w[2] -w[3] -w[4] -b[1] -b[2] 说明 序列最短长度(bp) 平均最大错误率 序列尾部最大错误率 滑动窗口长度(bp) 窗口内平均最大错误率 大滑动窗口内小滑动窗长度(bp) 窗口内平均最大错误率 序列头、尾检验长度(bp) 最大错误率 参数设置 50 0.03 0.03 30 0.03 10 0.1 4 0.03 c. Blastx：-m 0 –F F –e 1e-3 –b 5 –v 5

参数 -m -F

说明 输出格式 低复杂度序列是否过滤 参数设置 0 F 10

-e -b -v d. Newbler

参数 Use duplicate reads Extend low depth overlaps Minimus read length Reads limited to one contig 说明 拼接过程是否使用duplicate序列 低Depth区域是否延伸 Reads最短长度(bp) [20-45] 一个Reads是否只允许出现在一条Contig中 参数设置 Checked Checked 45 Checked 期望值 输出alignment的序列个数 输出 1e-3 5 5 e. Misa：1-10, 2-6, 3-5, 4-5, 5-5, 6-5, max_difference_between_2_SSRs=100

参数 1 2 3 4 5 6 Max_difference_between_2_SSRs 说明 单碱基连续重复次数 双碱基连续重复次数 3碱基连续重复次数 4碱基连续重复次数 5碱基连续重复次数 6碱基连续重复次数 两个SSR间最大间隔长度(bp) 参数设置 10 6 5 5 5 5 100 3.2 结果文件目录说明

目录 /assembly_result /database_annotation /SSR /SNP /GO /different_gene 说明 Newbler拼接结果文件 Swiss-Prot注释后结果文件 SSR分析结果文件 SNP分析结果文件 GO分析结果文件 差异基因分析结果 11

/tblastx Unigene与EST/mRNA比对结果 4. 参考文献

[1] Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., & Madden T.L. (2008) \

[2] http://sourceforge.net/projects/seqclean/

[3] LUCY2: an interactive DNA sequence quality trimming and vector removal tool Bioinformatics Advance Access published on May 6, 2004

[4] Huang, X. and Madan, A. (1999) CAP3: A DNA Sequence Assembly Program, Genome Research, 9: 868-877.

[5] http://pgrc.ipk-gatersleben.de/misa/

[6] ssahaSNP A Polymorphism Detection Tool on a Whole Genome Scale 2005 IEEE Computational Systems Bioinformatics Conference - Workshops, Vol. 0 (2005), pp. 251-254.

[7] Gene ontology: tool for the unification of biology. Nat. Genet.. May 2000;25(1):25-9. [8] Kanehisa, M., Goto, S., Furumichi, M., Tanabe, M., and Hirakawa, M.; KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res. 38, D355-D360 (2010).

[9] The Universal Protein Resource (UniProt) in 2010 Nucleic Acids Res. 38:D142-D148 (2010).

5. 附录（实验部分protocol）

5.1 RNA提取

*Invitrogen TRIzol Reagent

5.1.1 RNA precipitation

a.

(Optional) When precipitating RNA from small sample quantities (<106 cells or <10 mg tissue), add

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5–10 μg of RNase-free glycogen as a carrier to the aqueous phase. b.

Note: Glycogen is co-precipitated with the RNA, but does not inhibit first-strand synthesis at concentrations ≤4 mg/mL, and does not inhibit PCR. c.

Add 0.5 mL of 100% isopropanol to the aqueous phase, per 1 mL of TRIzol? Reagent used for homogenization. d. e. f.

Incubate at room temperature for 10 minutes. Centrifuge at 12,000 × g for 10 minutes at 4°C.

Note: The RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom of the tube. g.

Proceed to RNA wash.

5.1.2 RNA wash

a. b.

Remove the supernatant from the tube, leaving only the RNA pellet.

Wash the pellet, with 1 mL of 75% ethanol per 1 mL of TRIzol? Reagent used in the initial homogenization. c. d. e. f.

Note: The RNA can be stored in 75% ethanol at least 1 year at –20°C, or at least 1 week at 4°C. Vortex the sample briefly, then centrifuge the tube at 7500 × g for 5 minutes at 4°C. Discard the wash. Vacuum or air dry the RNA pellet for 5–10 minutes. Do not dry the pellet by vacuum centrifuge. Note: Do not allow the RNA to dry completely, because the pellet can lose solubility. Partially dissolved RNA samples have an A260/280 ratio <1.6. g.

Proceed to RNA resuspension.

5.1.3 RNA resuspension

a.

Resuspend the RNA pellet in RNase-free water or 0.5% SDS solution (20–50 μL) by passing the solution up and down several times through a pipette tip. b. c.

Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions. Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes. Proceed to downstream application, or store at –70°C.

d.

5.2反转录成cDNA

*Clonetech SMART

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5.2.1 First-strand cDNA synthesis, set up 2 tubes for 2ug polyA RNA, for each tube:

a.

add the following components to a nuclease-free 0.2ml PCR tube:

X ul Nuclease-free H2O 1-10ul

1ug polyA RNA

1ul dT15VN2 primer (50uM) 1ul 10mM dNTP

----------------------------------------------- 12ul Total

b. briefly mix, spin.. c. 65C, 5min. 4C 2min.

d.

add the following components: 4ul 5x First-Strand buffer 1ul 0.1M DTT 1ul RNaseOUT (40u/ul) 2ul

SuperScript III RT (200u/ul)

--------------------------------------------------- 20ul Total e. 50C, 1h f.

70C, 15min

g. 4C, 2 min, spin the content down

5.2.2 Second strand synthesis, for each tube:

a.

add the following component:

91ul Nuclease-free H2O 30ul 5X Second-Strand buffer 3ul 10mM dNTP

1ul E coli DNA ligase(10U/ul) 4ul E coli DNA polymerase(10U/ul) 1ul

E coli RNaseH (2 U/ul)

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------------------------------------------------------ 150ul Total

b. c. d. e. f. g. h. i.

briefly vortex mix, spin, remove content into 1.5ml Nuclease-free tube 16C, 2h

put the tube on ice, add 2ul T4 DNA polymerase(5U/ul) briefly vortex mix, spin 16C, 5 min place the tube on ice

add 10ul 0.5M EDTA pH8, to stop the reaction (optional) briefly vortex mix, spin

5.2.3 Phenol chloroform (Corrosive) isoamyl alcohol (PCI) and EtOH precipitation

NOTE: Extraction should be done in hood wearing safety goggles. Waste should be disposed of in a separate designated container.

Important Safety NOTE: Phenol is a corrosive agent which burns skin upon contact, practice extreme safety when performing the following steps.

a. b. c. d. e. f.

spin two Eppendorf phase-lock Light tubes (Green)

remove cDNA reaction content to phase-lock tube, transfer tubes to fume-hood. (lab#459) add 150ul PCI/tube, mix by shaking the tube vigorously for 15 second centrifuge @ 13000g RT, 5min

transfer top layer into 1.5ml clean Nuclease-free tube for each tube, add the following component:

15ul (1/10vol) 1.2ul

3M NaOAc, PH5.2

Glycogen (to help precipitate DNA)

EtOH

415ul (2.5vol)

g. h. i. j.

mix by inverting the tubes.

place the tubes in -70C for >1h (optional).

centrifuge @ 13000g RT for 20min. (prepare 2% agarose gel while waiting).

remove tubes from centrifuge gently, look for a white ~1 mm size pellet on the hinge side. This pellet should be present even if there were no DNA/RNA. If not present, check to see if all reagents were added. Spin again if needed to see the pellet.

k. l. m. n. o.

remove the supernatant with tip pointing away from the pellet, check to make sure the pellet remains after you remove the supernatant wash pellet with 500ul 70% EtOH centrifuge @ 13000g RT for 2min.

remove the supernatant, air dry @RT for 10min or SpeedVac dry for 5min

resuspend pellets of 2 tubes in total 15ul of nuclease-free H2O (sample can be stored @ -20C).

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5.2.4 Gel separation

a. b. c. d. e. f.

prepare 2% 1XTAE agarose gel 0.1 mg/ml Ethidium Bromide (Carcinogen) (need ~30min) save 2ul of sample (as a control) and run gel-purified sample together on Bioanalyzer add 2.5ul 5x loading dye (Qiagen) to remaining sample

pipette mix and load on gel, load 50bp & 100bp DNA ladder (Fermentas)

run gel @ 100V for ~10 min (to let the sample migrate from well in to gel quickly) then @50V for ~30min (cm to maximize separation of small molecular weight primer away from the cDNA)

image gel for 1 second (to minimize UV exposure) and save photos of both pre-cut and post-cut gels. cDNA are normally not visible @1 second exposure time, gel picture can be adjusted later so that cDNA are visible. The primer should run below 50bp. g. h.

cut gel slice from 100bp up to well using UV transluminator (make sure not to include the primer into your gel piece), place it in a 15 or 50ml falcon tube

Important! Minimize UV exposure to yourself (use long sleeves lab coats and face shield) and to the samples (use laminated paper to block UV to the samples not being cut). Turn off UV after cutting each sample. Transfer agar pieces to tubes with normal light. Review cut gel to make sure the right pieces of gel were transferred. i.

take another gel picture with 5 second exposure time. clean the gel and image area (gel slice can be stored @-20C)

j.

5.2.5 Gel extraction

a. b. c. d. e. f. g. h. i. j. k. l. m. n. o. p. q.

weight the gel slice add 3X volume of QC buffer

rotate the tube @37C or 50C incubator for 15 or 10min respectively, until no visible gel slice present add 1X volume of isopropanol, mix

the following steps can be performed by vacuum or centrifuge (13000g, 1min per) load the sample to Minelute column

applied the column to vacuum manifold or centrifuge, repeat this step 3-4 times if you have more than 750ul (load sample to another column if you have more than 3ml) apply 500ul QG buffer for each column, vacuum or centrifuge

apply 750ul PE buffer for each column, vacuum or centrifuge, repeat this step once place the column into emptied 2ml tube, centrifuge @13000g, 1min rotate tube 180C, centrifuge @13000g, 30second place the column to a nuclease-free 1.5ml tube add 12ul EB, wait 1min centrifuge @13000g, 1min.

use same EB solution to elute the other tube of the same sample (sample can then be stored @ -20C) determine cDNA concentration by Bioanalyzer (Bioanalyzer 7500 kit). cDNA should be >80ng/ul (total >1ug) if you start with 2ug PolyA RNA.

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5.2.6 Sonication

a. b. c. d. e. f. g. h. i. j.

use pump to circulate ice water to cool sonicator to 4C. dilute >1ug cDNA in 100ul TE in a 1.5 microfuge tube.

place tube in a rack in water bath that allows the bottom of the tube to touch the sonicator horn. make sure the liquid level of the sample is below the sonicator bath.

sonicate for 8 min at 1 min intervals with 1 min off between to allow sample to cool. spin 10sec.

Minelute purify cDNA, elute in 12ul

determine DNA concentration and size by Bioanalyzer DNA1000 kit. cDNA should be >80ng/ul (total >1ug) range 100-800bp.

gel purify cDNA between 100 to 800bp if needed (see step 4, 5 for details)

5.3 454 library preparation

*GS FLX Titanium General Library Preparation Kit, etc.

5.3.1 Fragmentation of RNA

a. b. c. d. e. f. g. h. i. j. k. l. m. n.

Start with 200 ng of sample RNA in a 200 μl PCR tube. Add Molecular Biology Grade Water to a fi nal volume of 19 μl.

Pipet 1 μl of this solution into a new 200 μl tube, and add 2 μl of Molecular Biology Grade Water. This sample will be compared to the fragmented RNA later on.

To the 18 μl of sample RNA, add 2 μl of RNA Fragmentation Solution, and vortex. Spin the tube for 2 seconds in a mini centrifuge.

Preheat the thermocycler to 70°C before placing the sample tube in. Heat the tube at 70°C for 30 seconds, with the heated lid on. Immediately, place the tube on ice.

Add 2 μl of 0.5 M EDTA, pH 8.0 and 28 μl of 10 mM Tris HCl, pH 7.5. Vortex and spin for 2 seconds in a mini centrifuge.

Add 80 μl of RNAClean reagent, containing SPRI beads.

Mix well by pipetting up and down 10 times, and incubate at room temperature for 10 minutes. Place the tube on a 96 ring Magnetic Particle Concentrator (MPC).

When the beads have completely pelleted on the side of the tube, carefully remove and discard all supernatant, without disturbing the beads.

Keeping the tube on the MPC, wash the beads three times, as follows, without disturbing the beads: i. ii. o. p. q. r.

Add 200 μl of 70% ethanol.

Completely remove and discard the ethanol.

Keeping the tube on the MPC, uncap the tube and air dry the pellet at room temperature for 3 minutes. Remove the tube from the MPC. Add 19 μl of 10 mM Tris.HCl, pH 7.5.

Vortex for 20 sec, spin for 2 seconds in a mini centrifuge, and pellet the beads on the MPC.

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s. t. u. v.

Without disturbing the beads, transfer the supernatant (~19 μl), containing the RNA, to a new 200 μl PCR tube. Place the tube on ice.

Pipet 1 μl of this solution in a 200 μl tube, and add 2 μl of Molecular Biology Grade Water. This sample will be compared to the one from step 3.

Run 1 μl of the fragmented (from step 19) and 1 μl of the non-fragmented RNA (from step 3) on an RNA 6000 Pico Chip on the Agilent 2100 Bioanalyzer to confi rm that the material has been fragmented Proceed with the fragmented sample (~17 μl).

5.3.2 Double-stranded cDNA Synthesis

Retrieve a cDNA Synthesis System Kit (Roche) and thaw the reagents before placing them on ice. Also thaw a tube of Roche Primer “random” 400 μM. 5.3.2.1 Denaturation of RNA a. b. c. d.

To the fragmented RNA, add 4 μl of the 400 μM Roche Primer “random”. Vortex for 10 seconds.

Spin for 2 seconds in a mini centrifuge.

Incubate the tube at 70°C for 10 minutes and immediately after, place on ice for 2 minutes.

5.3.2.2 First Strand cDNA Synthesis a.

With the tube on ice, add the following reagents: 8 μl vial 1 (5× RT-buffer AMV) 4 μl vial 3 (DTT, 0.1 M) 4 μl vial 7 (dNTPs, 10 mM)

1 μl vial 4 (Protector RNase Inhibitor, 25 U/μl) 2 μl vial 2 (AMV RT, 25 U/μl) 40 μl Total volume b. c. a.

Vortex gently for 2 seconds and spin for 2 seconds in a mini centrifuge.

Incubate the tube at 25°C for 10 minutes and then at 42°C for 60 minutes, and transfer on ice. To the 40 μl of sample, add the following reagents: 30 μl vial 9 (5× 2nd strand synthesis buffer) 72 μl vial 12 (redist water) 1.5 μl vial 7 (dNTPs, 10 mM)

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5.3.2.3 Second Strand cDNA Synthesis

6.5 ul vial 10 (2nd strand enzyme) 150 ul Total volume b. c. d. e. f.

Vortex 5 seconds and spin for 2 seconds in a mini centrifuge. Incubate the tube at 16°C for 2 hours.

Add 20 μl of vial 11 (T4 DNA Polymerase) and vortex gently for 5 seconds. Incubate at 16°C for 5 minutes.

Add 17 μl of 0.2 M EDTA, pH 8 to stop the reaction. Vortex 5 seconds and spin for 2 seconds in a mini centrifuge.

5.3.2.4 Double-stranded cDNA Purifi cation a. b. c. d. e. f.

Transfer the sample to a new 1.7 ml microcentrifuge tube. Add 300 μl of AMPure beads.

Vortex for 10 seconds and incubate at room temperature for 5 minutes. Place the tube on a Magnetic Particle Concentrator (MPC).

When the beads have completely pelleted on the side of the tube, carefully remove and discard all supernatant, without disturbing the beads.

Keeping the tube on the MPC, wash the beads three times, as follows, without disturbing the beads: a) b) g. h. i. j. k. l.

Add 800 μl of 70% ethanol.

Completely remove and discard the ethanol.

Keeping the tube on the MPC, uncap the tube and air dry the pellet at room temperature for 3 minutes. Remove the tube from the MPC.

Add 16 μl of 10 mM Tris-HCl, pH 7.5. Vortex for 20 seconds and spin for 2 seconds in a mini centrifuge. Place the tube on the MPC, wait for the beads to pellet on the wall of the tube and transfer the SUPERNATANT

(~16 μl), containing the double-stranded cDNA to a new 200 μl PCR tube, and place the tube on ice. We recommend processing the cDNA sample through to the end of the protocol. However, the cDNA sample can be stored over night at -80°C.

5.3.3 Fragment End Repair

Obtain a Rapid Library Prep kit. a.

In a 1.7 ml microcentrifuge tube, prepare the End Repair mix, as follows.

2.5 μl RL 10× Buffer 2.5 μl RL ATP 1 μl RL dNTP 1 μl RL T4 Polymerase 1 μl RL PNK

1 μl RL Taq Polymerase

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