ena-STUDY-Klinikum rechts der Isar, II. Medizinische Klinik-25-10-2017-12:50:03:166-570
Bulk 3-prime transcript end RNA-Sequencing of KrasG12D-driven mouse pancreatic cancer cell cultures derived from primary pancreatic tumors (n=38).
We generated low passage (<10) primary pancreatic cancer cell cultures from mice expressing mutant KrasG12D conditionally in the pancreas (PK mice, n=38). <br /> Total RNA was isolated with the RNeasy Mini kit (Qiagen) from 60-80% confluent 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 HiSeq1500 with 16 cycles for read 1 to decode sample barcodes and UMIs and 51 cycles on read 2 into the cDNA fragment. 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. Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to the ENSEMBL annotation release 75. Further analyses were performed with R version 3.2.2. Initial hierarchical clustering of samples was achieved using the pairwise sample correlation as distance measure for top 10% variable genes. Bootstrapping was performed to access cluster stability with the pvclust package v2.0 (Suzuki et al. [Bioinformatics. 2006 Jun 15;22(12):1540-2]). The 4 most prominent clusters were selected and differential expression between these clusters was calculated 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 DAVID 6.8 (Huang da et al. [Nat Protoc. 2009;4(1):44-57]) or the hypergeometric test as implemented on the “Molecular Signature Database” (MSigDB) v6.0 homepage (http://software.broadinstitute.org/gsea/msigdb/annotate). <br /> <br /> Barcodes (samples sequenced in technical replicates TR1/2): 16990-TR1-CTGCGG, 16990-TR2-GCCCTC, 16992-TR1-GGCCAC, 16992-TR2-GGTGGC, 2259-TR1-AAAACT, 2259-TR2-AATCTT, 3202-TR1-ATATAG, 3202-TR2-ATTTCA, 3250-TR1-GTTTAT, 3250-TR2-TAGTAA, 4072-TR1-TGTTTA, 4072-TR2-TTTACA, 4706-TR1-GCTAGA, 4706-TR2-GTACCG, 4900-TR1-CCCACG, 4900-TR2-CCTGGC, 5123-TR1-CGGTGG, 5123-TR2-GCACGG, 5320-TR1-GCTCGC, 5320-TR2-GGGGAC, 53578-TR1-AAGATT, 53578-TR2-ACTTAT, 53631-TR1-ATTACT, 53631-TR2-CTATAT, 53646-TR1-TAATGA, 53646-TR2-TATTGT, 53704-TR1-TTATTG, 53704-TR2-AGACCT, 5671-TR1-AAAGTT, 5671-TR2-AATTCT, 5748-TR1-ATCAAA, 5748-TR2-CAAATA, 6075-TR1-TAAAGT, 6075-TR2-TATAGA, 7662-TR1-TTAATC, 7662-TR2-TTTCTT, 8028-TR1-GGGATT, 8028-TR2-ACCGGC, 8182-TR1-CCCCGT, 8182-TR2-CGAGGC, 8248-TR1-CCAACC, 8248-TR2-AGGGGC, 8296-TR1-CGTGGG, 8296-TR2-GCCAGG, 8305-TR1-GGAGCC, 8305-TR2-GGGTCG, 8349-TR1-AAATTG, 8349-TR2-ACAATA, 8442-TR1-ATGAAT, 8442-TR2-CATTAT, 8513-TR1-TAAGAT, 8513-TR2-TATGAA, 8570-TR1-TTAGTT, 8570-TR2-TTTTGA, 8661-TR1-GTTCGA, 8661-TR2-ACGGGG, 9091-TR1-CCGTCG, 9091-TR2-CGCTGC, 9203-TR1-GAGCGG, 9203-TR2-GCGAGC, 9591-TR1-CCCTGG, 9591-TR2-CGCCCT, B590-TR1-TCTGCA, B590-TR2-AGCGGG, R1035-TR1-CCGGAC, R1035-TR2-CGCGCA, S134-TR1-GACCGC, S134-TR2-GCCGTG, S302-TR1-GGCGTC, S302-TR2-GTGCGC, S411-TR1-AATACA, S411-TR2-AGATTA, S559-TR1-ATTCTA, S559-TR2-CTTTAA, S821-TR1-TACTAT, S821-TR2-TCATAT, S914-TR1-TTGAAA, S914-TR2-CACCAA
We generated low passage (<10) primary pancreatic cancer cell cultures from mice expressing mutant KrasG12D conditionally in the pancreas (PK mice, n=38). <br /> Total RNA was isolated with the RNeasy Mini kit (Qiagen) from 60-80% confluent 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 HiSeq1500 with 16 cycles for read 1 to decode sample barcodes and UMIs and 51 cycles on read 2 into the cDNA fragment. 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. Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to the ENSEMBL annotation release 75. Further analyses were performed with R version 3.2.2. Initial hierarchical clustering of samples was achieved using the pairwise sample correlation as distance measure for top 10% variable genes. Bootstrapping was performed to access cluster stability with the pvclust package v2.0 (Suzuki et al. [Bioinformatics. 2006 Jun 15;22(12):1540-2]). The 4 most prominent clusters were selected and differential expression between these clusters was calculated 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 DAVID 6.8 (Huang da et al. [Nat Protoc. 2009;4(1):44-57]) or the hypergeometric test as implemented on the “Molecular Signature Database” (MSigDB) v6.0 homepage (http://software.broadinstitute.org/gsea/msigdb/annotate). <br /> <br /> Barcodes (samples sequenced in technical replicates TR1/2): 16990-TR1-CTGCGG, 16990-TR2-GCCCTC, 16992-TR1-GGCCAC, 16992-TR2-GGTGGC, 2259-TR1-AAAACT, 2259-TR2-AATCTT, 3202-TR1-ATATAG, 3202-TR2-ATTTCA, 3250-TR1-GTTTAT, 3250-TR2-TAGTAA, 4072-TR1-TGTTTA, 4072-TR2-TTTACA, 4706-TR1-GCTAGA, 4706-TR2-GTACCG, 4900-TR1-CCCACG, 4900-TR2-CCTGGC, 5123-TR1-CGGTGG, 5123-TR2-GCACGG, 5320-TR1-GCTCGC, 5320-TR2-GGGGAC, 53578-TR1-AAGATT, 53578-TR2-ACTTAT, 53631-TR1-ATTACT, 53631-TR2-CTATAT, 53646-TR1-TAATGA, 53646-TR2-TATTGT, 53704-TR1-TTATTG, 53704-TR2-AGACCT, 5671-TR1-AAAGTT, 5671-TR2-AATTCT, 5748-TR1-ATCAAA, 5748-TR2-CAAATA, 6075-TR1-TAAAGT, 6075-TR2-TATAGA, 7662-TR1-TTAATC, 7662-TR2-TTTCTT, 8028-TR1-GGGATT, 8028-TR2-ACCGGC, 8182-TR1-CCCCGT, 8182-TR2-CGAGGC, 8248-TR1-CCAACC, 8248-TR2-AGGGGC, 8296-TR1-CGTGGG, 8296-TR2-GCCAGG, 8305-TR1-GGAGCC, 8305-TR2-GGGTCG, 8349-TR1-AAATTG, 8349-TR2-ACAATA, 8442-TR1-ATGAAT, 8442-TR2-CATTAT, 8513-TR1-TAAGAT, 8513-TR2-TATGAA, 8570-TR1-TTAGTT, 8570-TR2-TTTTGA, 8661-TR1-GTTCGA, 8661-TR2-ACGGGG, 9091-TR1-CCGTCG, 9091-TR2-CGCTGC, 9203-TR1-GAGCGG, 9203-TR2-GCGAGC, 9591-TR1-CCCTGG, 9591-TR2-CGCCCT, B590-TR1-TCTGCA, B590-TR2-AGCGGG, R1035-TR1-CCGGAC, R1035-TR2-CGCGCA, S134-TR1-GACCGC, S134-TR2-GCCGTG, S302-TR1-GGCGTC, S302-TR2-GTGCGC, S411-TR1-AATACA, S411-TR2-AGATTA, S559-TR1-ATTCTA, S559-TR2-CTTTAA, S821-TR1-TACTAT, S821-TR2-TCATAT, S914-TR1-TTGAAA, S914-TR2-CACCAA