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Illumina Sequencing Library Construction from ChIP DNA
将ChIP方法获取的DNA用来构建Illumina测序文库   

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Abstract

The Illumina sequencing platform is very popular among next-generation sequencing platforms. However, the DNA sequencing library construction kit provided by Illumina is considerably expensive. The protocol described here can be used to construct high-quality sequencing libraries from chromatin immunoprecipitated DNA. It uses key reagents from third-party vendors and greatly reduces the cost in library construction for Illumina sequencing.

Materials and Reagents

  1. QIAquick PCR purification kit (QIAGEN, catalog number: 28104 )
  2. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
  3. MinElute PCR purification kit (QIAGEN, catalog number: 28004 ), store the columns at 4 °C.
  4. Gibco UltraPure water (Life Technologies, Gibco®, catalog number: 10977-015 )
  5. End-it DNA End repair kit (Epicentre®, catalog number: ER0720 )
  6. Klenow fragment (3’ ≥ 5’ exo minus) (New England Biolabs, catalog number: M0212S )
  7. 100 mM dATP (Life Technologies, Invitrogen™, catalog number: 10216-018 or VWR International, catalog number: PAU1201 )
  8. LigaFast ligation kit (Promega corporation, catalog number: M8221 )
  9. Ethanol
  10. Elution buffer
  11. End repair buffer
  12. Klenow buffer
  13. Cyan/orange loading buffer
  14. 2% E-gel, precast agarose gel (Life Technologies, Invitrogen™) (can be replaced by house made 2% agarose gel)
  15. Phusion HF PCR master mix (New England Biolabs, catalog number: F531S or F531L )
  16. Illumina Adapter oligo mix from Illumina sequencing kit, but can be replaced by house made oligo mix using fast denaturation and slow reannealing as described in reference 1. The adapter oligo sequences are:
    5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
    5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
  17. Illumina PCR primers 1.1 and 2.1 from Illumina sequencing kit, but can be replaced by ordinary synthesized oligos with the following sequences:
    Illum1.1:
    5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCC
    GATCT
    Illum2.1:
    5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT

Equipment

  1. BECKMAN centrifuges and rotor (Beckman Coulter)
  2. PCR thermocycler (PerkinElmer or F. Hoffmann-La Roche)
  3. NanoDrop Micro-Volume UV-Vis Spectrophotometer

Procedure

  1. Purification from ethanol precipitated ChIP DNA. Use QIAquick PCR purification kit, and elute in 50 μl elution buffer. Use 34 μl for library construction and the rest for qPCR verification.
  2. Input DNA needs to be diluted before making library. After purification, measure the concentration of input DNA by Nanodrop. If it reads 20 ng/μl, dilute 5 fold, if close to 40 ng/μl dilute 10 fold.
  3. End-repair:
    Mix the following components in a 1.5 ml Eppendorf tube:
    34 μl purified ChIP DNA (or add Gibco water up to 34 μl)
    5 μl 10x end repair buffer
    5 μl 2.5 mM dNTP mix
    5 μl 10 mM ATP
    1 μl end-repair enzyme mix
    50 μl total
    Incubate for 45 min at room temperature (RT).
    Purify using QIAquick PCR purification column, elute in 34 μl.
  4. Addition of ‘A’ base to the 3’ ends:
    Mixing the following components in a 1.5 ml Eppendorf tube:
    34 μl ChIP DNA from step 3
    5 μl Klenow buffer = NEB buffer 2
    10 μl 1 mM dATP
    1 μl Klenow fragment (3’-5’ exo minus)
    50 μl total reaction volume
    (1 mM dATP is diluted from 100 mM dATP stock and aliquoted in 25 μl, freeze-thaw only once)
    Incubate 30 min at 37 °C.
    Purify using MinElute PCR purification column, elute in 12 μl.
  5. Adaptor ligation:
    Mix the following components in a 1.5 ml Eppendorf tube:
    11 μl ChIP DNA from step 4
    15 μl DNA ligase buffer
    1 μl 1:20 diluted adaptor oligo
    3 μl DNA ligase
    30 μl total
    (If doing multiplexing, make sure to add in each reaction with different adaptor, and label clearly.)
    Incubate 15 min at RT.
  6. Gel selection to get rid of excessive adaptors:
    Dilute cyan/orange loading buffer 10 fold, add 6 μl to the 30 μl reactions from step 5, load in two wells of an E-gel. Also load 20 μl 10-fold diluted 50 bp ladder. Run E-gel for 20 min.
    Cut between 150 and 450 bp. Purify the DNA using QIAquick gel extraction kit, elute in 30 μl buffer.
  7. PCR with Illumina primers:
    1:1 dilute Illumina PCR primers 1.1 and 2.1 with Gibco water.
    Mixing the following components in PCR stripe tubes:
    30 μl ChIP DNA from step 4
    28 μl Phusion PCR mastermix
    1 μl diluted primer 1.1
    1 μl diluted primer 2.1
    60 μl total reaction volume
    Run PCR cycle:
    98 °C 30 sec
    98 °C 10 sec
    65 °C 30 sec
    72 °C 30 sec
    GOTO step 2 for 15 times
    72 °C 5 min
    4 °C hold.
  8. Size selection on 2% agarose gel:
    Add 1 μl undiluted cyan/orange loading dye to the PCR reaction, load all in 3 wells on E-gel, run for 30 min alongside with 20 μl 1:10 diluted 50 bp ladder and 20 μl 1:10 diluted 100 bp ladder.
    Cut between 150 and 450 bp. Take pictures before and after the slice is excised. Make sure to avoid the ~100 bp adaptor band. A good library should have a smear centering at ~200 bp. Strong band at ~100 bp indicates over amplification of adaptors and the library may not be good enough quality.
    Purify the DNA using QIAquick gel extraction kit and elute in 30 μl buffer.
  9. Measure DNA concentration:
    Use NanoDrop to measure the DNA concentration. A good library should be relatively concentrated (e.g., > 10 ng/μl).

Acknowledgments

The protocol has been tested and optimized by different researchers in the Snyder lab, Stanford University (Lefrancois et al., 2009).

References

  1. Lefrancois, P., Euskirchen, G. M., Auerbach, R. K., Rozowsky, J., Gibson, T., Yellman, C. M., Gerstein, M. and Snyder, M. (2009). Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing. BMC Genomics 10: 37.
  2. Lefrancois, P., Zheng, W. and Snyder, M. (2010). ChIP-Seq using high-throughput DNA sequencing for genome-wide identification of transcription factor binding sites. Methods Enzymol 470: 77-104.

简介

Illumina测序平台在下一代测序平台中非常流行。 然而,Illumina提供的DNA测序文库构建试剂盒相当昂贵。 这里描述的协议可以用于从染色质免疫沉淀的DNA构建高质量的测序文库。 它使用来自第三方供应商的关键试剂,大大降低了Illumina测序文库构建的成本。

材料和试剂

  1. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
  2. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  3. MinElute PCR纯化试剂盒(QIAGEN,目录号:28004),将柱子保存在4℃。
  4. Gibco UltraPure水(Life Technologies,Gibco ,目录号:10977-015)
  5. 末端DNA末端修复试剂盒(Epicentre ,目录号:ER0720)
  6. Klenow片段(3'≥5'外切)(New England Biolabs,目录号:M0212S)
  7. 100mM dATP(Life Technologies,Invitrogen TM,目录号:10216-018或VWR International,目录号:PAU1201)
  8. LigaFast连接试剂盒(Promega corporation,目录号:M8221)
  9. 乙醇
  10. 洗脱缓冲液
  11. 结束修复缓冲区
  12. Klenow缓冲区
  13. 青色/橙色装载缓冲区
  14. 2%E-gel,预制琼脂糖凝胶(Life Technologies,Invitrogen TM)(可以用自制的2%琼脂糖凝胶代替)
  15. Phusion HF PCR主混合物(New England Biolabs,目录号:F531S或F531L)
  16. 来自Illumina测序试剂盒的Illumina Adaptor寡核苷酸混合物,但是可以使用如参考文献1中所述的快速变性和缓慢再退火的自制寡核苷酸混合物替换。接头寡核苷酸序列是:
    5'P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
    5'ACACTCTTTCCCTACACGACGCTCTTCCGATCT
  17. 来自Illumina测序试剂盒的Illumina PCR引物1.1和2.1,但可以用具有以下序列的普通合成寡核苷酸代替:
    Illum1.1:
    5'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCC
    GATCT
    Illum2.1:
    5'CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT

设备

  1. BECKMAN离心机和转子(Beckman Coulter)
  2. PCR热循环仪(PerkinElmer或F.Hoffmann-La Roche)
  3. NanoDrop微量紫外可见分光光度计

程序

  1. 从乙醇沉淀的ChIP DNA纯化。 使用QIAquick PCR纯化试剂盒,并在50μl洗脱缓冲液中洗脱。 使用34μl用于文库构建,其余用于qPCR验证
  2. 输入DNA需要在制作文库之前稀释。 纯化后,通过Nanodrop测量输入DNA的浓度。 如果读数为20 ng /μl,稀释5倍,如果接近40 ng /μl稀释10倍。
  3. 端修:
    将以下组分在1.5 ml Eppendorf管中混合:
    34μl纯化的ChIP DNA(或加入最多34μl的Gibco水)
    5μl10x末端修复缓冲液
    5μl2.5mM dNTP混合物
    5μl10 mM ATP
    1μl末端修复酶混合物
    总共50μl
    在室温(RT)下孵育45分钟 使用QIAquick PCR纯化柱纯化,在34μl中洗脱
  4. 在3'末端加上'A'碱基:
    将以下组分在1.5ml Eppendorf管中混合:
    34μl来自步骤3的ChIP DNA
    5μlKlenow缓冲液= NEB缓冲液2
    10μl1 mM dATP
    1μlKlenow片段(3'-5'外切负)
    50μl总反应体积
    (1mM dATP从100mM dATP储液稀释并等分为25μl,仅冻融一次)
    在37℃孵育30分钟。
    使用MinElute PCR纯化柱纯化,在12μl中洗脱
  5. 适配器连接:
    将以下组分在1.5 ml Eppendorf管中混合:
    11μl来自步骤4的ChIP DNA
    15μlDNA连接酶缓冲液
    1μl1:20稀释的接头寡聚体
    3μlDNA连接酶
    总共30μl
    (如果进行多路复用,请确保在每个反应中添加不同的适配器,并清楚标记。)
    在室温下孵育15分钟。
  6. 凝胶选择,以摆脱过多的适配器:
    将青色/橙色加载缓冲液稀释10倍,向来自步骤5的30μl反应物中加入6μl,加载到E-gel的两个孔中。 也加载20μl10倍稀释的50 bp梯。 运行E凝胶20分钟。
    在150和450 bp之间切割。 使用QIAquick凝胶提取试剂盒纯化DNA,在30μl缓冲液中洗脱
  7. 用Illumina引物进行PCR:
    用Gibco水稀释1:1稀释的Illumina PCR引物1.1和2.1。
    在PCR条管中混合以下组分:
    30μl来自步骤4的ChIP DNA
    28μlPhusion PCR mastermix
    1μl稀释引物1.1
    1μl稀释引物2.1
    总反应体积为60微升
    运行PCR循环:
    98°C 30秒
    98℃10秒
    65°C 30秒
    72°C 30秒
    GOTO步骤2 15次
    72℃5分钟
    4°C保持
  8. 在2%琼脂糖凝胶上的大小选择:
    加入1微升未稀释的青色/橙色加载染料到PCR反应,加载所有在3孔在E-gel,运行30分钟与20微升1:10稀释50 bp梯和20微升1:10稀释100 bp梯。
    在150和450 bp之间切割。 在切片切除前后拍照。 确保避免〜100 bp适配器带。 一个好的图书馆应该有一个以〜200 bp为中心的涂片。 在〜100bp的强谱带表示适配器的过扩增,并且文库可能不够好质量 使用QIAquick凝胶提取试剂盒纯化DNA,并在30μl缓冲液中洗脱
  9. 测量DNA浓度:
    使用NanoDrop测量DNA浓度。 好的文库应该相对集中(例如,<10ng /μl)。

致谢

该协议已经由斯坦福大学的Snyder实验室中的不同研究者测试和优化(Lefrancois等人,2009)。

参考文献

  1. Lefrancois,P.,Euskirchen,G.M.,Auerbach,R.K.,Rozowsky,J.,Gibson,T.,Yellman,C.M.,Gerstein,M.and Snyder,M.(2009)。 使用多重短读DNA测序的高效酵母ChIP-Seq。 BMC Genomics 10:37.
  2. Lefrancois,P.,Zheng,W。和Snyder,M。(2010)。 ChIP-Seq使用高通量DNA测序在转录因子结合位点的全基因组鉴定。/a> Methods Enzymol 470:77-104。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zheng, W. (2012). Illumina Sequencing Library Construction from ChIP DNA. Bio-protocol 2(4): e91. DOI: 10.21769/BioProtoc.91.
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