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RNA-Seq Library Generation from Rare Human Cells Isolated by FACS
采用FACS法分离稀有人体细胞及其RNA测序文库的构建   

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Abstract

High throughput RNA Sequencing has revolutionized transcriptome analyses. However, most available protocols require micrograms of RNA rendering this technique not feasible for analyzing small numbers of cells, including precious rare cell types isolated from human tissues or organs. Here, we used an RNA Amplification System and describe a method for preparing RNA sense-strand cDNA libraries compatible with an Illumina sequencing platform starting from limited numbers of human fetal germ cells as well as human embryonic stem cells (hESCs) isolated using Fluorescence Activated Cell Sorting (FACS). With this protocol we generated seven RNA-Seq libraries starting from 4,000 germ cells sorted from fetal ovaries (n = 2) and fetal testes (n = 2) at 16-16.5 weeks of development and 4,000 sorted hESCs (n = 3). We predict that multiplexed libraries can also be generated by replacing the single-plex 3’ adapter used here with a multiplexing compatible 3’ adapter and indexed PCR primers.

Keywords: PGC(PGC), Human(人类), Germ-cell(生殖细胞), RNA-Seq(RNA测序)

Materials and Reagents

  1. RNeasy Micro Kit (QIAGEN, catalog number: 74004 )
  2. WT-Ovation Pico RNA Amplification System (Nugen, catalog number: 3300 )
  3. WT-Ovation Exon Module (Nugen, catalog number: 2000 )
  4. Qubit dsDNA HS Assay Kit (Life Technologies, Invitrogen™, catalog number: Q32851 )
  5. Tris-EDTA pH 8.0, molecular biology grade (Life Technologies, Ambion®, catalog number: AM9849 )
  6. TruSeq DNA Sample Preparation Kit (Illumina, catalog number: FC-121-2001 )
  7. MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
  8. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
  9. Certified Low Range Ultra Agarose (Bio-Rad Laboratories, catalog number: 161-3107 )

Equipment

  1. BD FACSAria Fluorescence activated cell sorter
  2. NanoDrop 1000
  3. PCR Thermal Cycler
  4. Sonic Dismembrator 550 (Thermo Fisher Scientificc)
  5. Microtip (Misonix Inc, catalog number: 419 )
  6. Fluorometer
  7. Agarose gel running apparatus and UV-light transilluminator
  8. Illumina HiSeq 2000


    Figure 1. Overview of the protocol and main reagents used

Procedure

  1. Sort 4,000 cells directly in 75 μl of RLT buffer (Qiagen RNeasy Micro Kit) using BD FACSAria cell sorter.
  2. Isolate total RNA from sorted cells using the RNeasy Micro Kit according to manufacturer’s protocol without the addition of β-mercaptoethanol to RLT buffer. Perform on column DNA digestion for 15 min according the manufacturer’s instructions using the DNase that is provided with the RNeasy Micro Kit and elute in 14 μl of RNase/DNase free water. Yield of total RNA using a NanoDrop 1000 ranges between 30-80 ng.
  3. Amplify RNA and generate cDNA with complementary sequences to the original mRNA using the WT-Ovation Pico RNA Amplification System according to manufacturer’s instruction. Expected yield of amplified cDNA measured on a NanoDrop 1000 is between 5-10 μg. A quality control step can be included at this point by performing Real-Time PCR for known transcripts that are expected to be present in the amplified cDNA pool.
  4. Generate sense-strand cDNA targets from 3 μg of complementary sequence cDNA using the WT-Ovation Exon Module kit according to manufacturer’s instructions.
  5. Sonicate 3 μg of sense-strand cDNA that is diluted to a final volume of 400 μl Tris-EDTA pH 8.0 buffer to generate cDNA fragments in the 200-500 bp range. Sonication with Sonic Dismembrator 550 and a 419 Microtip is performed on ice with six pulses of 20 sec each (magnitude setting of 3) and a 60 sec rest interval.
  6. Quantify sense-strand sonicated cDNA on a fluorometer using the Qubit HS dsRNA assay kit according to manufacturers’ instructions and aliquot 30 ng for generating the Sequencing library.
  7. Perform End Repair (Reagents are part of the TruSeq DNA Sample Preparation Kit)
    Sense-strand cDNA (30 ng)
    30 μl                        
    Water
    10 μl
    T4 DNA Ligase Buffer with 10 mM ATP
    5 μl
    10 mM dNTP mix
    2 μl
    T4 DNA Polymerase (3 U μl-1)
    1 μl
    Klenow DNA Polymerase (1 U μl-1 diluted with water)
    1 μl
    T4 PNK (10 U μl-1)
    1 μl  
    20 °C 30 min on a thermal cycler
  8. Purify using the MinElute PCR Purification Kit according to manufacturer’s instructions and elute in 34 μl of RNase/DNase free water. Use all eluted cDNA in following step.
  9. Adenylate 3’ ends (Reagents are part of the TruSeq DNA Sample Preparation Kit)
    cDNA
    34 μl     
    10x Klenow buffer
    5 μl
    1 mM dATP
    10 μl
    Exo-Klenow (5 U μl-1)
    1 μl
    37 °C 30 min on a thermal cycler
  10. Purify using the MinElute PCR Purification Kit according to manufacturer’s instructions. Elute with 10 μl of RNase/DNase free water.
  11. Ligate paired-end adapters (reagents are part of the TruSeq DNA Sample Preparation Kit)
    cDNA
    10 μl                 
    2x Ligase buffer
    15 μl
    Adapter oligo mix (1/10 dilution in water)
    1 μl
    DNA ligase
    4 μl
    Room temperature 15 min
  12. Purify the ligation reaction using the MinElute PCR Purification Kit according to manufacturer’s instructions.
  13. Size select the cDNA library by running the purified PCR product through a 1.5% Agarose Gel, cutting a smear in the range of 150-300 bp, purifying using the QIAquick Gel Extraction Kit according to manufacturer’s instructions and eluting with 36 μl of RNase/DNase free water. Note that DNA concentration is very low therefore there is no visible DNA band on the gel.
  14. PCR amplification of the adapter modified DNA fragments (Reagents are part of the TruSeq DNA Sample Preparation Kit)
    DNA
    36 μl              
    5x Phusion buffer
    10 μl
    10 mM dNTPs
    1.5 μl
    PCR primer 1.1
    1 μl
    PCR primer 1.2
    1 μl
    Phusion DNA Polymerase (2 U μl-1)
    0.5 μl
    Use the following protocol on a thermal cycler
    1. 98 °C 30 sec
    2. 18 cycles of
          98 °C 10 sec
          65 °C 30 sec
          72 °C 30 sec
    3. 72 °C 5 min
    4. 4 °C hold
  1. Validate library by running the amplified DNA through a 1.5% Agarose Gel. The majority of the amplified cDNA should appear between the 150-300 bp range. Primer dimers and adapters appear at the 30-60 bp range.
  2. Purify library by gel extracting the DNA that appears in the 150-300 bp range using the QIAquick Gel Extraction Kit according to manufacturer’s instructions and eluting with 15 μl RNase/DNase free water (Figure 2). Quantify library final yield on a fluorometer using the Qubit HS dsDNA assay according to manufacturer’s instructions. The libraries generated using this protocol yielded between 200-700 ng.


    Figure 2. Example of gel extracted band alongside 100 bp ladder. Adapters visible around 30-60 bp. 

  3. A quality control step can be included at this point by performing Real-Time PCR for known transcripts that are expected to be present in the generated library. If a quality control step was performed at step 3, before generating the library, the results should be similar.
  4. Run the cDNA library on an Illunina HiSeq 2000. Alignment statistics for all samples analyzed as well as correlation scores on the 3 replicates of hESC RNA-Seq libraries are shown in Tables 1 and 2.

    Table 1. Alignment statistics
    Sample                         
    Total reads                   
    Uniquely aligned reads (% total)
    Testis 16 wk
    116,399,186
    35,497,900
    30.5     
    Testis 16.5 wk
    153,982,474
    37,907,565
    24.6 
    Ovary 16 wk
    102,837,931
    44,784,216
    43.5 
    Ovary 16.5 wk
    211,659,065
    93,766,963
    44.3
    hESCs 1
    161,037,803
    39,339,764
    24.4
    hESCs 2
    133,355,463
    38,571,046
    28.9
    hESCs 3
    104,997,235
    37,164,448
    35.4

    Table 2. Correlation scores for hESC RNA-Seq libraries

    hESCs 1
    hESCs 2
    hESCs 3
    hESCs 1
    1
    0.96
    0.96
    hESCs 2
    0.97
    1
    0.97
    hESCs 3
    0.96
    0.97
    1

Acknowledgments

A.T.C. is supported by funds from the NIH/NICHD 2 R01 HD058047.

References

  1. Gkountela, S., Li, Z., Vincent, J. J., Zhang, K. X., Chen, A., Pellegrini, M. and Clark, A. T. (2013). The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation. Nat Cell Biol 15(1): 113-122.

简介

高通量RNA测序革新了转录组分析。 然而,大多数可用的协议需要微克RNA,使得这种技术不可能用于分析少量的细胞,包括从人体组织或器官分离的珍贵的稀有细胞类型。 在这里,我们使用RNA扩增系统,并描述了一种制备RNA有义链cDNA文库的方法,其与Illumina测序平台相容,从有限数目的人胎儿生殖细胞以及使用荧光活化细胞分离的人胚胎干细胞(hESC) 排序(FACS)。 使用这个协议,我们生成七个RNA Seq库开始从4,000生殖细胞从胎儿卵巢(n = 2)和胎儿睾丸(n = 2)在16-16.5周的发展和4,000分选hESCs(n = 3)排序。 我们预测多重文库也可以通过用多重兼容的3'衔接子和索引的PCR引物替换这里使用的单重3'衔接子来产生。

关键字:PGC, 人类, 生殖细胞, RNA测序

材料和试剂

  1. RNeasy Micro Kit(QIAGEN,目录号:74004)
  2. WT-Ovation Pico RNA扩增系统(Nugen,目录号:3300)
  3. WT-Ovation Exon Module(Nugen,目录号:2000)
  4. Qubit dsDNA HS测定试剂盒(Life Technologies,Invitrogen TM,目录号:Q32851)
  5. Tris-EDTA pH 8.0,分子生物学级(Life Technologies,Ambion ,目录号:AM9849)
  6. TruSeq DNA样品制备试剂盒(Illumina,目录号:FC-121-2001)
  7. MinElute PCR纯化试剂盒(QIAGEN,目录号:28004)
  8. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  9. 认证的低范围超琼脂糖(Bio-Rad Laboratories,目录号:161-3107)

设备

  1. BD FACSAria荧光激活细胞分选机
  2. NanoDrop 1000
  3. PCR热循环仪
  4. Sonic Dismembrator 550(Thermo Fisher Scientificc)
  5. Microtip(Misonix Inc,目录号:419)
  6. 荧光计
  7. 琼脂糖凝胶电泳仪和紫外光透射仪
  8. Illumina HiSeq 2000


    图1.使用的协议和主要试剂概述

程序

  1. 使用BD FACSAria细胞分选仪,在75μlRLT缓冲液(Qiagen RNeasy Micro Kit)中直接分选4,000个细胞。
  2. 使用RNeasy Micro Kit根据制造商的方案从分选的细胞中分离总RNA,而不向RLT缓冲液中加入β-巯基乙醇。 根据制造商的说明,使用RNeasy Micro Kit提供的DNase在柱DNA上消化15分钟,并在14μl的RNase/DNase游离水中洗脱。 使用NanoDrop 1000的总RNA产量范围为30-80ng。
  3. 扩增RNA并使用WT-Ovation Pico RNA扩增系统根据制造商的说明产生具有与原始mRNA互补的序列的cDNA。在NanoDrop 1000上测量的扩增cDNA的预期产量在5-10μg之间。此时可以包括质量控制步骤,通过对预期存在于扩增cDNA库中的已知转录物进行实时PCR。
  4. 使用WT-Ovation Exon Module试剂盒根据制造商的说明书从3μg互补序列cDNA产生有义链cDNA靶。
  5. 超声处理3微克有义链cDNA,其被稀释至终体积为400μlTris-EDTA pH 8.0缓冲液以产生200-500bp范围的cDNA片段。使用Sonic Dismembrator 550和419 Microtip的超声处理在冰上用六个脉冲(每个20秒)(幅度设置为3)和60秒的休息间隔进行。
  6. 使用Qubit HS dsRNA测定试剂盒根据制造商的说明在荧光计上定量有义链超声处理的cDNA,并等分30ng以产生测序文库。
  7. 执行末端修复(试剂是TruSeq DNA样品制备试剂盒的一部分)
    有义链cDNA(30ng)
    30μl                       

    10微升
    T4 DNA连接酶缓冲液和10mM ATP 5微升
    10mM dNTP mix
    2微升
    T4 DNA聚合酶(3Uμl -1
    1微升
    Klenow DNA聚合酶(1 Uμl 用水稀释) 1微升
    T4 PNK(10 Uμl -1
    1μl  
    在热循环仪上20℃30分钟
  8. 根据制造商的说明书使用MinElute PCR纯化试剂盒纯化,并在34μl的RNase/DNase游离水中洗脱。 在后续步骤中使用所有洗脱的cDNA。
  9. 腺苷酸3'端(试剂是TruSeq DNA样品制备试剂盒的一部分)
    cDNA
    34μl     
    10x Klenow缓冲区
    5微升
    1 mM dATP
    10微升
    Exo-Klenow(5Uμl -1
    1微升
    在热循环仪上37℃30分钟
  10. 根据制造商的说明使用MinElute PCR纯化试剂盒进行纯化。 用10μl无RNase/DNase的水洗脱。
  11. Ligation配对末端衔接头(试剂是TruSeq DNA样品制备试剂盒的一部分)
    cDNA
    10μl                 
    2x连接酶缓冲液
    15微升
    接头寡核苷酸混合物(1/10在水中稀释)
    1微升
    DNA连接酶
    4微升
    室温15分钟
  12. 使用MinElute PCR纯化试剂盒根据制造商的说明纯化连接反应
  13. 通过使纯化的PCR产物通过1.5%琼脂糖凝胶运行,切割150-300bp范围内的涂片,选择cDNA文库,使用QIAquick凝胶提取试剂盒根据制造商的说明书纯化,并用36μlRNA酶/DNA酶 自由水。 注意,DNA浓度很低,因此凝胶上没有可见的DNA条带。
  14. 接头修饰的DNA片段的PCR扩增(试剂是TruSeq DNA样品制备试剂盒的一部分)
    DNA
    36微升              
    5x Phusion缓冲区
    10微升
    10 mM dNTPs
    1.5μl
    PCR引物1.1
    1微升
    PCR引物1.2
    1微升
    Phusion DNA聚合酶(2Uμl -1
    0.5μl
    在热循环仪
    上使用以下协议
    1. 98°C 30秒
    2. 18个周期的
           98°C 10秒
           65°C 30 sec
           72°C 30秒
    3. 72℃5分钟
    4. 4°C保持
  1. 通过运行扩增的DNA通过1.5%琼脂糖凝胶验证文库。大多数扩增的cDNA应当出现在150-300bp范围之间。引物二聚体和衔接子出现在30-60bp范围。
  2. 通过凝胶提取出现在150-300 bp范围内的DNA使用QIAquick凝胶提取试剂盒根据制造商的说明书,用15μlRNase/DNase游离水(图2)洗脱纯化文库。使用Qubit HS dsDNA测定根据制造商的说明书在荧光计上定量文库最终产率。使用此协议产生的文库产生200-700ng。


    图2.沿着100bp梯度的凝胶提取条带的实施例。 适配器可见大约30-60 bp。

  3. 此时可以包括质量控制步骤,通过对预期存在于所产生的文库中的已知转录物进行实时PCR。如果在步骤3执行质量控制步骤,则在生成库之前,结果应该类似
  4. 在Illunina HiSeq 2000上运行cDNA文库。所有样品的比对统计以及hESC RNA-Seq文库的3个重复的相关性评分显示在表1和2中。

    表1.对齐统计信息
    示例                        
    总阅读                 
    唯一对齐的读数(总共%)
    睾丸16周

    35,497,900
    30.5     
    睾丸16.5周


    24.6 
    卵巢16周


    43.5 
    卵巢16.5周


    44.3
    hESC 1


    24.4
    hESCs 2


    28.9
    hESCs 3

    37,164,448
    35.4

    表2. hESC RNA-Seq图书馆的相关性分数

    hESCs 1
    hES Cs 2
    hESCs 3
    hES Cs 1
    1
    0.96
    0.96
    hESCs 2
    0.97
    1
    0.97
    hESCs 3
    0.96
    0.97
    1

致谢

A.T.C. 由NIH/NICHD 2 R01 HD058047的资金支持。

参考文献

  1. Gkountela,S.,Li,Z.,Vincent,J.J.,Zhang,K.X.,Chen,A.,Pellegrini,M.and Clark,A.T。 cKIT +人类原始生殖细胞的个体发育证明是人类种系重编程,印迹擦除 和体外分化。 Nat Cell Biol 15(1):113-122
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gkountela, S. and Clark, A. T. (2013). RNA-Seq Library Generation from Rare Human Cells Isolated by FACS. Bio-protocol 3(12): e791. DOI: 10.21769/BioProtoc.791.
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