RNA-SEQ assay for wild type and CRISPR induced endoglin knockout human pulmonary artery smooth muscle cells (PASMC)

The goal of this study is to investigate the differential transcripted genes affected by CRISPR induced endoglin knockout in PASMC cells. Total RNA was purified from NTC or ENG-/- PASMC cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quality and concentration were assessed with Agilent Tapestation 200 (Agilent Technologies) and Qubit 2.0 (ThermoFisher Scientific). ~250-500 ng RNA were used for library construction. The NGS libraries were constructed using the KAPA Stranded mRNA-Seq Kits (KapaBioSystems). mRNA was captured using magnetic oligo-dT beads and 1st strand cDNA was synthesized using random priming. In order to preserve strand-specificity, 2nd strand synthesis, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), was marked by dUTP incorporation. cDNA framents were A-tailed by adding dAMP to the 3'-ends of the dscDNA library fragments. dsDNA Illumina TruSeq "forked” adapters 3'-dTMP overhangs were then ligated to A-tailed library insert fragments. Each of the six libraries were ligated with a unique Truseq 6bp barcode. Library fragments were amplified using the KAPA HiFi HotStart polymerase. The strand marked with dUTP was not amplified, allowing strand-specific sequencing. Fragment length and library quality was assessed on a 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies). Libraries were diluted to 10nM and pooled at equimolar ratios. The pool was then diluted to 2nM and denatured in NaOH following Illumina recommendations. 10pM of denatured library pool was loaded in one HiSeq lane and flowcell was clustered on the Illumina C-bot. 5% PhiX control was spiked-in. The flowcell was sequenced on a HiSeq 2500 V4 chemistry with 50bp Single read protocol. Data was demultiplexed and Fastq files were generated using BcptoFastq 1.8.4 script provided by Illumina.

本研究旨在探究CRISPR诱导的内皮糖蛋白（endoglin）敲除对肺动脉平滑肌细胞（PASMC）中差异转录基因的影响。实验采用RNeasy迷你总RNA纯化试剂盒（Qiagen，德国希尔德）从非靶向对照（NTC）或ENG敲除（ENG-/-）的PASMC细胞中纯化总RNA。通过安捷伦Tapestation 200（安捷伦科技）与Qubit 2.0（赛默飞世尔科技）分别评估RNA的质量与浓度。取约250~500 ng RNA用于文库构建。本研究使用KAPA链特异性mRNA测序文库构建试剂盒（KapaBioSystems）构建下一代测序（NGS）文库。首先利用寡聚dT磁珠捕获mRNA，随后采用随机引物法合成第一链cDNA。为保留链特异性，在将cDNA:RNA杂交双链转换为双链cDNA（dscDNA）的第二链合成步骤中，通过掺入dUTP对合成链进行标记。随后在dscDNA文库片段的3'末端添加dAMP以完成cDNA片段的A尾加尾。将带有3'-dTMP突出端的双链DNA Illumina TruSeq“分叉”接头连接至加A尾的文库插入片段。六个文库均分别连接有独特的TruSeq 6bp条形码。使用KAPA HiFi HotStart DNA聚合酶对文库片段进行扩增，由于掺入dUTP标记的链无法被扩增，因此实现了链特异性测序。采用高灵敏度DNA试剂盒（安捷伦科技）在2100生物分析仪上评估文库片段的长度与质量。将文库稀释至10 nM，并以等摩尔比例混合。随后将混合文库稀释至2 nM，按照Illumina官方推荐流程使用氢氧化钠进行变性。取10 pM变性后的文库混合液上样至一个HiSeq测序通道，并在Illumina C-bot上完成流动槽测序簇生成。同时掺入5%的PhiX对照文库。使用HiSeq 2500 V4测序化学试剂，采用50bp单端读长测序方案完成测序。数据通过Illumina提供的BcptoFastq 1.8.4脚本进行解多路复用，并生成Fastq格式文件。