This kit is highly recommended for users who:

• Would like to identify and quantify full-length transcripts
• Are looking for a faster and simpler method for cDNA synthesis: ~210 minutes library prep + variable time for PCR
• Want to explore isoforms, splice variants and fusion transcripts using full-length cDNAs
• Wish to start from total RNA or have a low starting amount of RNA
• Would like to generate high amounts of cDNA data

This protocol outlines a targeted cDNA sequencing method using the cDNA-PCR Sequencing Kit (SQK-PCS111) and a user-defined sequence-specific primer. You can swap out the cDNA RT Adapter (CRTA) and design your own primer to target a specific RNA sequence during reverse transcription. During the strand-switching step, a UMI is incorporated before the double-stranded cDNA is amplified by PCR using primers containing rapid attachment chemistry. The rapid sequencing adapters are then added to the amplified sample.

Steps in the sequencing workflow:

Prepare for your experiment You will need to:

• Extract your RNA, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
• Ensure you have your sequencing kit, the correct equipment and third-party reagents.
• Download the software for acquiring and analysing your data.
• Check your flow cell to ensure it has enough pores for a good sequencing run.

**Library preparation** You will need to:

• Perform reverse transcription with sequence-specific primer.
• Using the strand-switching protocol, prepare full-length cDNAs from your RNA sample.
• Amplify the samples by PCR, adding rapid attachment primers during the PCR step.
• Ligate sequencing adapters to the PCR products.
• Prime the flow cell, and load your cDNA library into the flow cell.

Sequencing and analysis You will need to:

• Start a sequencing run using the MinKNOW software, which will collect raw data from the device and will basecall the reads.
• (Optional) Start the EPI2ME software and select a workflow for further analysis, e.g. metagenomic analysis or drug resistance mapping

The cDNA RT Adapter (CRTA) supplied in the cDNA-PCR Sequencing Kit (SQK-PCS111) is designed to ligate to poly(A) tailed RNAs. However, you can design your own primer to target a specific RNA sequence (for example, to only sequence 16S rRNA) for subsequent cDNA sequencing.

To perform this type of targeting, order the oligo below, replacing the [sequence-specific] region with ~22 bases complementary to the 3' end of your target RNA sequence. Please note that the exact number of bases may need to be optimised depending on the sequence targeted.

5' - ACTTGCCTGTCGCTCTATCTTC - [sequence-specific] - 3'

We recommend starting with a stock concentration of 2 μM. However, you may find it useful to perform a titration of different primer concentrations. All primers should be HPLC purified.

选择符合质量和浓度要求的起始RNA至关重要的。使用过少或过多的RNA，或者质量较差的RNA（如，高度碎片化、或含有化学污染物的RNA）都会影响文库制备。

有关如何对DNA样品进行质控，请参考起始DNA/RNA质控。

有关使用RNA作为起始材料的更多信息，请参阅以下链接：

• 使用大肠杆菌多聚腺苷酸聚合酶为RNA转录本添加poly-A尾
• RNA污染物
• RNA稳定性
• RNA完整值（RIN）
• 多聚腺苷酸化RNA分子的富集

您亦可在纳米孔社区的DNA/RNA Handling 页面找到上述文件。

Oxford Nanopore Technologies推荐您使用本实验指南中提及的所有第三方试剂，并已对其加以验证。我们尚未对其它替代试剂进行测试。

我们建议您按制造商说明准备待用的第三方试剂.

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Strand Switching Primer II	SSPII	Violet	1	20 µl
RT Primer	RTP	Yellow	1	10 µl
cDNA RT Adapter	CRTA	Amber	1	10 µl
Rapid Adapter T	RAP T	Green	1	10 µl
Annealing Buffer	AB	Orange	1	10 µl
cDNA Primer	cPRM	White cap, grey label	1	40 µl
Elution Buffer	EB	Black	1	500 µl
Short Fragment Buffer	SFB	Clear	1	1,800 µl
Sequencing Buffer II	SBII	Red	1	500 µl
Loading Beads II	LBII	Pink	1	360 µl
Loading Solution	LS	White cap, pink label	1	400 µl
Flush Buffer	FB	Blue	6	1,170 µl
Flush Tether	FLT	White cap, purple label	1	200 µl

请为MinION Mk1B配备一台高规格的计算机或笔记本电脑，以适配数据采集的速度。您可以在MinION Mk1B的IT配置要求文件中了解更多。

MinION Mk1C是一款集计算功能和触控屏幕于一体的便携式测序分析仪，它无需依赖任何额外设备，即可生成并分析纳米孔测序数据。您可以在 MinION Mk1C的IT配置要求文件中了解更多。

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.

For instructions on how to run the MinKNOW software, please refer to the MinKNOW protocol.

EPI2ME (optional)

The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome, barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-basecalling.

For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to the EPI2ME Platform protocol.

我们强烈建议您在开始测序实验前，对测序芯片的活性纳米孔数进行质检。质检需在您收到MinION /GridION /PremethION测序芯片三个月之内进行，或者在您收到Flongle测序芯片四周内进行。Oxford Nanopore Technologies会对活性孔数量少于以下标准的芯片进行替换** ：

测序芯片	芯片上的活性孔数确保不少于
Flongle 测序芯片	50
MinION/GridION 测序芯片	800
PromethION 测序芯片	5000

** 请注意：自收到之日起，芯片须一直贮存于Oxford Nanopore Technologies推荐的条件下。且质检结果须在质检后的两天内递交给我们。请您按照 测序芯片质检文档中的说明进行芯片质检。

We recommend using the Loading Beads II (LBII) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Loading Solution (LS) instead of water. Note: some customers have noticed that viscous libraries can be loaded more easily when not using Loading Beads II.

在完成测序芯片的加样后，您即可在MinKNOW中启动测序实验。MinKNOW 软件负责仪器控制、数据采集以及实时碱基识别。有关设置和使用 MinKNOW 的详细信息，请参阅MinKNOW 实验指南。

您可以通过多种方式使用并设置MinKNOW：

• 在直接或远程连接到测序设备的计算机上。
• 直接在 GridION、MinION Mk1C 或 PromethION 24/48 测序设备上。

有关在测序设备上使用 MinKNOW 的更多信息，请参阅相应设备的用户手册：

• MinION Mk1B 用户手册
• MinION Mk1C 用户手册
• GridION 用户手册

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在MinKNOW中启动测序：

1. 在 "开始 "（Start）页面上，选择 __开始测序__（Start Sequencing)。

2. 输入实验详情：例如实验名称，测序芯片位置及样本ID。

3. 在"试剂盒"页面上，选择建库试剂盒。

4. 配置测序实验参数，或保持“运行选项”和“分析”页面中的默认设置。

请注意： 如果在设置运行参数时关闭了碱基识别，您可在实验结束后，在MinKNOW中运行线下碱基识别。详情请参阅MinKNOW实验指南。

5. 在“输出”页面中，设置输出参数或保持默认设置。

6. 单击 "参数确认" 页面上的 开始 启动测序。

当于MinKNOW上完成测序后，您可按照“测序芯片的重复利用及回收”一节中的说明重复使用或返还测序芯片。

完成测序和碱基识别后，即可进行数据分析。有关碱基识别和后续分析选项的详细信息，请参阅数据分析文档。

在下游分析部分，我们将概述更多用于数据分析的选项。

您可以选择以下几个途径来进一步分析经过碱基识别的数据：

1. EPI2ME 工作流程

Oxford Nanopore Technologies通过EPI2ME Labs平台提供了一系列针对高阶数据分析的生物信息学教程和工作流程。上述资源汇总于纳米孔社区的 EPI2ME Labs 板块。该平台通过描述性文字、生物信息学代码和示例数据，具象化地展示出我们的研究和应用团队发布在 GitHub 上的工作流程。

2. 科研分析工具

Oxford Nanopore Technologies的研发部门开发了许多分析工具，您可在Oxford Nanopore的 GitHub 资料库中找到。这些工具面向有一定经验的用户，并包含如何安装和运行软件的说明。工具以源代码形式提供，因此我们仅提供有限的技术支持。

3. 纳米孔社区用户开发的分析工具

如果以上工具仍无法为您提供解决研究问题的分析方法，请参考资源中心的生物信息学板块。该板块汇总了许多由纳米孔社区成员开发、且在Github上开源的、针对纳米孔数据的生信分析工具。请注意，Oxford Nanopore Technologies不为这些工具提供支持，也不能保证它们与测序所用的最新的化学试剂/软件配置兼容。

我们还在 Nanopore 社区的“Support”板块 提供了常见问题解答（FAQ）。

如果以下方案仍无法解决您的问题，请通过电邮(support@nanoporetech.com)）或微信公众号在线支持（NanoporeSupport）联系我们。

我们还在 Nanopore 社区的“Support”板块 提供了常见问题解答（FAQ）。

如果以下方案仍无法解决您的问题，请通过电邮(support@nanoporetech.com)）或微信公众号在线支持（NanoporeSupport）联系我们。