ena-STUDY-Klinikum rechts der Isar, II. Medizinische Klinik-08-11-2017-14:14:41:127-413
Bulk 3-prime transcript end RNA-Sequencing of epithelial/mesenchymal primary pancreatic cancer cell cultures enriched from a CRISPR/Cas9-edited, KrasG12D-driven mouse model of pancreatic cancer.
Multiplexed gene editing of tumor suppressor genes using CRISPR/Cas9 in the pancreas of a KrasG12D-driven mouse model of pancreatic cancer was performed as described earlier by Maresch et al. (Nat Commun. 2016 Feb 26;7:10770). Primary cultures of induced mPDACs were isolated and monitored for the simultaneous presence of epithelial and mesenchymal phenotypes. Enrichment of epithelial and mesenchymal cell morphologies was achieved by differential exposition times to trypsin (Thermo Fisher Scientific Inc.). Short-term incubation (2-3 min) induced detachment of mesenchymal cells, while epithelial colonies remained adherent. Both cell fractions were subsequently grown to 80% confluency in new flasks. This process was repeated for 3-6 times until homogenous epithelial and mesenchymal cell fractions were enriched. Total RNA was isolated with the RNeasy Mini kit (Qiagen) from 60-80% confluent epithelial/mesenchymal pancreatic cancer cells cultured at 37°C with 5% CO2 in DMEM medium supplemented with 10% fetal calf serum without P/S. RNA was reversely transcribed using oligo-dT primers decorated with sample barcodes, unique molecular identifiers (UMIs) and adapters (Integrated DNA Technologies). cDNA from all samples was pooled and un-incorporated primers digested using ExonucleaseI (New England Biolabs). Next, the cDNA pool was amplified with KAPA HiFi ReadyMix (KAPA Biosystems). To obtain sequencing libraries, 0.8ng of cDNA was tagmented and 3' ends amplified with the Nextera XT Kit (Illumina) using a specific primer for the adapter on the 3'-end. The library was paired-end sequenced on a NextSeq550 with 71 cycles on read 1 into the cDNA fragment and 21 cycles for read 2 to decode sample barcodes and UMIs. Data was processed using the published Drop-seq pipeline (v1.0) (Macosko et al. [Cell. 2015 May 21;161(5):1202-1214]) to generate sample- and gene-wise UMI tables. Alignment to the human reference genome (GRCh 38p10). Transcript and gene definitions were used according to the ENSEMBL annotation release 87. Further analyses were performed with R version 3.2.2. Group comparisons (epithelial vs. mesenchymal) were conducted with DESeq2 ([Genome Biol. 2014;15(12):550]). A gene was considered to be differentially regulated if the absolute log2-foldchange was above 0.8. Gene set enrichment testing was performed with hypergeometric test as implemented on the “Molecular Signature Database” (MSigDB) v6.0 homepage (http://software.broadinstitute.org/gsea/msigdb/annotate).
Multiplexed gene editing of tumor suppressor genes using CRISPR/Cas9 in the pancreas of a KrasG12D-driven mouse model of pancreatic cancer was performed as described earlier by Maresch et al. (Nat Commun. 2016 Feb 26;7:10770). Primary cultures of induced mPDACs were isolated and monitored for the simultaneous presence of epithelial and mesenchymal phenotypes. Enrichment of epithelial and mesenchymal cell morphologies was achieved by differential exposition times to trypsin (Thermo Fisher Scientific Inc.). Short-term incubation (2-3 min) induced detachment of mesenchymal cells, while epithelial colonies remained adherent. Both cell fractions were subsequently grown to 80% confluency in new flasks. This process was repeated for 3-6 times until homogenous epithelial and mesenchymal cell fractions were enriched. Total RNA was isolated with the RNeasy Mini kit (Qiagen) from 60-80% confluent epithelial/mesenchymal pancreatic cancer cells cultured at 37°C with 5% CO2 in DMEM medium supplemented with 10% fetal calf serum without P/S. RNA was reversely transcribed using oligo-dT primers decorated with sample barcodes, unique molecular identifiers (UMIs) and adapters (Integrated DNA Technologies). cDNA from all samples was pooled and un-incorporated primers digested using ExonucleaseI (New England Biolabs). Next, the cDNA pool was amplified with KAPA HiFi ReadyMix (KAPA Biosystems). To obtain sequencing libraries, 0.8ng of cDNA was tagmented and 3' ends amplified with the Nextera XT Kit (Illumina) using a specific primer for the adapter on the 3'-end. The library was paired-end sequenced on a NextSeq550 with 71 cycles on read 1 into the cDNA fragment and 21 cycles for read 2 to decode sample barcodes and UMIs. Data was processed using the published Drop-seq pipeline (v1.0) (Macosko et al. [Cell. 2015 May 21;161(5):1202-1214]) to generate sample- and gene-wise UMI tables. Alignment to the human reference genome (GRCh 38p10). Transcript and gene definitions were used according to the ENSEMBL annotation release 87. Further analyses were performed with R version 3.2.2. Group comparisons (epithelial vs. mesenchymal) were conducted with DESeq2 ([Genome Biol. 2014;15(12):550]). A gene was considered to be differentially regulated if the absolute log2-foldchange was above 0.8. Gene set enrichment testing was performed with hypergeometric test as implemented on the “Molecular Signature Database” (MSigDB) v6.0 homepage (http://software.broadinstitute.org/gsea/msigdb/annotate).