ena-STUDY-Klinikum rechts der Isar, II. Medizinische Klinik-25-10-2017-15:20:25:428-593
Bulk 3-prime transcript end RNA-Sequencing of KRASG12D- and GFP-overexpressing human pancreatic cancer cell lines.
cDNA of oncogenic KRASG12D (CCDS 8702.1, 35G>A) and GFP were cloned into the pINDUCER20 (Meerbrey et al. [Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3665-70]) lentiviral vector. Lentivirus was produced using HEK293FT cells transfected with standard virus packaging plasmids and respective pINDUCER20 vectors by following manufacturer's recommendations. Virus-containing supernatant was pooled 48h and 72h post transfection, concentrated by polyethylene glycol 6000 precipitation (Kutner et al. [Nat Protoc. 2009;4(4):495-505]). 1x105 HUPT3 and PANC0327 hPDAC cells were transduced in presence of 1µg/mL polybrene and selected with puromycin antibiotic. Target gene expression was induced by the addition of 100ng/µL doxycycline into P/S-free culturing medium. Total RNA was isolated with the RNeasy Mini kit (Qiagen) from 60-80% confluent, doxycycline-induced human pancreatic cancer cell lines 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. Alignments 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 (KRASG12D vs GFP) 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 and the adjusted P-value was =0.05. 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).
cDNA of oncogenic KRASG12D (CCDS 8702.1, 35G>A) and GFP were cloned into the pINDUCER20 (Meerbrey et al. [Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3665-70]) lentiviral vector. Lentivirus was produced using HEK293FT cells transfected with standard virus packaging plasmids and respective pINDUCER20 vectors by following manufacturer's recommendations. Virus-containing supernatant was pooled 48h and 72h post transfection, concentrated by polyethylene glycol 6000 precipitation (Kutner et al. [Nat Protoc. 2009;4(4):495-505]). 1x105 HUPT3 and PANC0327 hPDAC cells were transduced in presence of 1µg/mL polybrene and selected with puromycin antibiotic. Target gene expression was induced by the addition of 100ng/µL doxycycline into P/S-free culturing medium. Total RNA was isolated with the RNeasy Mini kit (Qiagen) from 60-80% confluent, doxycycline-induced human pancreatic cancer cell lines 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. Alignments 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 (KRASG12D vs GFP) 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 and the adjusted P-value was =0.05. 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).