Poly(A)+ RNA-seq from H226 cells expressing doxycycline-inducible Control (non-targeting) and p63-targeting shRNAs

To determine the impact of ?Np63a knockdown on steady-state mRNA levels, we performed poly(A)-enriched RNA-seq analysis of lung squamous cell carcinoma line H226 (inducible shControl and shp63) in the presence of 1µg/mL doxycycline to induce shRNA expression. Overall design: Poly(A)+ RNA for two independent biological replicates was purified from H226 cells (inducible shControl and shp63) incubated treated for six days with 1 µg/mL doxycycline. a TruSeq Stranded mRNA Library Prep Kit (Illumina). Libraries were sequenced on an Illumina HiSeq 2000 system at the University of Colorado Cancer Center Genomics and Microarray Core facility. Reads were aligned (TopHat2) to the Human reference genome (GRCh37/hg19) and gene-level counts (HTseq-count) were used for differential expression analysis (DESeq2).

为探究ΔNp63a基因敲低对细胞稳态mRNA水平的影响，我们以携带诱导型对照短发卡RNA（shControl）与靶向p63短发卡RNA（shp63）的肺鳞状细胞癌细胞系H226为研究对象，在1μg/mL多西环素诱导短发卡RNA（shRNA）表达的条件下，开展了聚腺苷酸化富集RNA测序（poly(A)-enriched RNA-seq）分析。整体实验设计如下：将上述两种H226细胞经1μg/mL多西环素培养处理6天后，提取两个独立生物学重复的Poly(A)+ RNA；采用TruSeq链特异性mRNA文库制备试剂盒（Illumina）构建测序文库；随后在科罗拉多大学癌症中心基因组与微阵列核心实验室的Illumina HiSeq 2000测序平台上完成测序。测序reads通过TopHat2比对至人类参考基因组GRCh37/hg19，利用HTseq-count获取基因水平计数矩阵，并通过DESeq2进行差异表达分析。