1.gene expression of the organoids and their parental tissues and tumors by RNA-seq 2.the gene mutations detected in tumor organoids and the parental tumor tissue
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RNA-seq analysis Standard RNA-seq analyses were performed by Sangon Biotech (Shanghai) Co., Ltd. China (for hiPSC-derived organoids) and GENEWIZ (Suzhou) Co., Ltd. China (for patient tissues and patient-derived organoids). The hiPSCs, tissues and organoids after 7 days in culture were send to GENEWIZ (Suzhou, China) or Sangon Biotech (Shanghai, China) to perform the standard RNA-seq analysis. Total RNA of each sample was extracted using the TRIzol Reagent (Invitrogen) /RNeasy Mini Kit (Qiagen). 1 μg total RNA with RIN value above 6.5 was used for library preparation. Next generation sequencing library preparations were constructed according to the manufacturer’s protocol. Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument according to manufacturer’s instructions. Sequencing was carried out using a 2× 150bp paired-end (PE) configuration; image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument. Differential expression analysis was performed using the DESeq2 Bioconductor package, a model based on the negative binomial distribution. The estimates of dispersion and logarithmic fold changes incorporate data-driven prior distributions. Padj of genes were set <0.05 to detect differential expressed ones. KEGG (Kyoto Encyclopedia of Genes and Genomes) is a collection of databases dealing with genomes, biological pathways, diseases, drugs, and chemical substances (http://en.wikipedia.org/wiki/KEGG). We used scripts in house to enrich significant differential expression gene in KEGG pathways. Whole-exome sequencing (WES) analysis Whole exome sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. China (for hiPSC-derived organoids) and GENEWIZ (Suzhou) Co., Ltd. China (for patient tumor tissues and patient-derived organoids). The tumor tissues and the organoids cultured for 7 days were sent to GENEWIZ (Suzhou, China) to perform the whole exome sequencing by Illumina HiSeq. Primary analysis was performed by built-in software, HiSeq Control Software (HCS), RTA 2.3 plus, and demultiplexing was performed by bcl2fastq 2.17. The raw data of exome sequencing were analyzed by bioinformatics analysts of GENEWIZ Inc. The GATK haplotypecaller or samtools was used to call SNV and the variants were annotated by Annovar (Version11Feb2016).