DNBelab C4单细胞RNA-Seq文库制备流程基于Drop-Seq技术原理，匹配自主研发的多磁珠识别技术，包括两种捕获磁珠，分别为大磁珠和小磁珠。大磁珠表面修饰有长、短两种捕获序列，数目多达10的7次方。长捕获序列用于捕获mRNA，短捕获序列用于捕获小磁珠释放的序列。小磁珠对来自同一个液滴中的多个大磁珠进行相关性数据合并。大小磁珠的联合应用优化了实验体系，提高了细胞捕获效率将细胞（核）悬液用0.4%台盼蓝染色，在显微镜下镜检并统计活率，活率大于80%即可进入建库环节。文库制备使用DNBelab C 系列高通量单细胞RNA文库制备试剂盒套装 V3.0（MGI，China）。依次将准备好的单细胞悬液、油、beads加入C4 scRNA载片与装置中，将细胞（核）悬液制备成液滴，在液滴中完成细胞裂解及磁珠捕获mRNA的过程。随后，液滴被真空泵击碎，释放出mrna -头复合物。cDNA合成在合适的温度下。然后扩增和纯化cDNA和Oligo产物。cDNA产物和oligo产物质检其浓度和片段分布，以确保在良好的范围内。构建Oligo文库，然后进行扩增，加index和纯化以进行循环化。cDNA片段化、末端修复和加“A”形成cDNA产物。将cDNA在合适的温度下连接到接头上固定一段时间，然后纯化。用PCR扩增带接头的cDNA，然后纯化。cDNA和Oligo产物分别变性为单链。变性为单链后，配制环化反应体系，并设置反应程序，得到单链环形产物，并消化未被环化的线性DNA分子。通过滚环扩增复制单链环状DNA分子，生成包含多个DNA拷贝的DNA纳米球(DNA nanoball, DNB)。然后使用高强度DNA纳米芯片技术将足够质量的DNA加到芯片上的网状小孔内，并通过组合探针-锚定聚合技术(cPAS)进行测序。cDNA文库用PE47+100的测序模式，Oligo文库则用PE32+42的测序模式进行测序。DNBelab C4 adopts the self-developed multi magnetic bead recognition technology of large and small size based on a Drop-seq. The average surface modification of a single magnetic bead is 10^7 long-type and short-type capture sequences. Long-type capture sequences are used to capture mRNA while short-type for capturing the mRNAs released by small-size magnetic beads. Small-size magnetic beads integrate the data from multi large-size magnetic beads of the same droplet. Combined application of small- and large-size magnetic beads optimises experimental system and improves cell capture efficiency. The cell or nuclei suspension is stained with 0.4% trypan blue to assess cell viability under microscopic observation. Cells with greater than 80% viability are qualified for library construction process.Library construction is performed by using DNBelab C Series High-throughput Single-cell RNA Library Preparation Set V3.0 (MGI,China). The single-cell suspension, oil, beads are added into C4 scRNA slide and instrument in order. Following this, the droplets are broken by vacuum pump, releasing the mRNA-bead complexes.cDNA synthesis is conducted at the suitable temperature. Then the cDNA and Oligo products are amplified and purified. The products are subjected to concentration and size distribution test to make sure in a good range.The Oligo library is constructed and then subjected to amplification, index ligation and purification for circularization.The cDNA is fragmentated, end repair and A tailing to form cDNA products. Then the cDNA is ligate to adaptor at the suitable temperature for a fixed period of time and then purified. The cDNA with adaptor is amplified via PCR and then purified. The cDNA and Oligo products are respectively denatured into single stranded. The reaction system and program for circularization are respectively configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested. Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).cDNA library was sequenced by PE 47+100 and Oligo library using PE 32+42.
